Antigen-binding proteins with increased fcrn binding

ABSTRACT

The present invention provides antigen binding proteins which bind specifically to TNF-alpha. For example novel variants of anti-TNF antibodies such as adalimumab which show increased binding to the FcRn receptor or increased half life compared to adalimumab. Also provided are compositions comprising the antigen binding proteins and uses of such compositions in treatment of disorders and disease.

FIELD

The invention relates to novel variants of anti-TNF antibodies.

BACKGROUND

Antibodies are heteromultimeric glycoproteins comprising at least twoheavy and two light chains. Aside from IgM, intact antibodies areusually heterotetrameric glycoproteins of approximately 150 Kda,composed of two identical light (L) chains and two identical heavy (H)chains. Each heavy chain has at one end a variable domain (VH) followedby a number of constant regions. Each light chain has a variable domain(VL) and a constant region at its other end; the constant region of thelight chain is aligned with the first constant region of the heavy chainand the light chain variable domain is aligned with the variable domainof the heavy chain. Depending on the amino acid sequence of the constantregion of their heavy chains, human antibodies can be assigned to fivedifferent classes, IgA, IgD, IgE, IgG and IgM. IgG and IgA can befurther subdivided into subclasses, IgG1, IgG2, IgG3 and IgG4; and IgA1and IgA2. The variable domain of the antibody confers bindingspecificity upon the antibody with certain regions displaying particularvariability called complementarity determining regions (CDRs). The moreconserved portions of the variable region are called Framework regions(FR). The variable domains of intact heavy and light chains eachcomprise four FR connected by three CDRs. The constant regions are notdirectly involved in the binding of the antibody to the antigen butexhibit various effector functions such as participation in antibodydependent cell-mediated cytotoxicity (ADCC), phagocytosis via binding toFcγ receptor, half-life/clearance rate via neonatal Fc receptor (FcRn)and complement dependent cytotoxicity via the C1q component of thecomplement cascade. The nature of the structure of an IgG antibody issuch that there are two antigen-binding sites, both of which arespecific for the same epitope. They are therefore, monospecific.

In adult mammals, FcRn, also known as the neonatal Fc receptor, plays akey role in maintaining serum antibody levels by acting as a protectivereceptor that binds and salvages antibodies of the IgG isotype fromdegradation. IgG molecules are endocytosed by endothelial cells, and ifthey bind to FcRn, are recycled out into circulation. In contrast, IgGmolecules that do not bind to FcRn enter the cells and are targeted tothe lysosomal pathway where they are degraded.

The neonatal FcRn receptor is believed to be involved in both antibodyclearance and the transcytosis across tissues (see Junghans R. P (1997)Immunol. Res 16. 29-57 and Ghetie et al (2000) Annu. Rev. Immunol. 18,739-766).

WO 9734631 discloses a composition comprising a mutant IgG moleculehaving increased serum half-life and at least one amino acidsubstitution in the Fc-hinge region. Amino acid substitution at one ormore of the amino acids selected from number 252, 254, 256, 309, 311 or315 in the CH2 domain or 433 or 434 in the CH3 domain is disclosed.

WO 00/42072 discloses a polypeptide comprising a variant Fc region withaltered FcRn binding affinity, which polypeptide comprises an amino acidmodification at any one or more of amino acid positions 238, 252, 253,254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317,340, 356, 360, 362, 376, 378, 380, 386, 388, 400, 413, 415, 424, 433,434, 435, 436, 439, and 447 of the Fc region.

WO 02/060919 discloses a modified IgG comprising an IgG constant domaincomprising amino acid modifications at one or more of positions 251,253, 255, 285-290, 308-314, 385-389, and 428-435.

WO 2004035752 discloses a modified antibody of class IgG wherein atleast one amino acid residue from the heavy chain constant regionselected from the group consisting of amino acid residues 250, 314, and428 is different from that present in an unmodified class IgG antibody.

Shields et al. (2001, J Biol Chem; 276:6591-604) used alanine scanningmutagenesis to alter residues in the Fc region of a human IgG1 antibodyand then assessed the binding to human FcRn. Positions that effectivelyabrogated binding to FcRn when changed to alanine include 1253, S254,H435, and Y436. Other positions showed a less pronounced reduction inbinding as follows: E233-G236, R255, K288, L309, S415, and H433. Severalamino acid positions exhibited an improvement in FcRn binding whenchanged to alanine.

Dall'Acqua et al. (2002, J Immunol.; 169:5171-80) described randommutagenesis and screening of human IgG1 hinge-Fc fragment phage displaylibraries against mouse FcRn. They disclosed random mutagenesis ofpositions 251, 252, 254-256, 308, 309, 311, 312, 314, 385-387, 389, 428,433, 434, and 436.

WO2006130834 discloses modified IgG comprising an IgG comprising an IgGconstant domain comprising amino acid modifications at one or morepositions of 252, 254, 256, 433, 434 and 436.

Therefore, modification of Fc domains of IgG antibodies has beendiscussed as a means of increasing the serum half-life of therapeuticantibodies. However, numerous such modifications have been suggestedwith varying and sometimes contradictory results in differentantibodies.

The administration of antigen binding proteins as therapeutics requiresinjections with a prescribed frequency relating to the clearance andhalf-life characteristics of the protein.

Adalimumab is a monoclonal antibody against TNF-alpha which is used fortreatment of rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis, and Crohn's disease. It is produced by recombinant DNAtechnology using a mammalian cell expression system. It consists of 330amino acids and has a molecular weight of approximately 148 kilodaltons.See U.S. Pat. No. 6,090,382. At doses of 0.5 mg/kg (˜40 mg), clearancefor adalimumab is said to range from 11 to 15 ml/hour, the distributionvolume (V_(ss)) ranges from 5 to 6 litres and the mean terminal phasehalf-life was approximately two weeks (Summary of ProductCharacteristics available from www.medicines.org.uk). These half lifeand clearance properties mean that currently adalimumab needs to beadministered once every two weeks. In some patients depending on diseaseit may be necessary to administer a loading dose such as for example inpsoriasis patients. This dosage may differ from the maintenance dose.

SUMMARY OF INVENTION

In one aspect, the invention relates to an antigen binding protein whichspecifically binds to TNF-alpha comprising CDRH1 (SEQ ID NO: 27), CDRH2(SEQ ID NO: 28), CDRH3 (SEQ ID No: 29), CDRL1 (SEQ ID NO: 30), CDRL2(SEQ ID NO: 31), and CDRL3 (SEQ ID NO: 32) or variants thereof whereinsaid variants may contain 1, 2, 3 or 4 amino acid substitutions,insertions or deletions as compared to CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, or CDRL3; and a neonatal Fc receptor (FcRn) binding portion of ahuman IgG1 constant domain comprising one of more amino acidsubstitutions relative to the human IgG1 constant domain, wherein theantigen binding protein has an increased FcRn binding affinity at pH 6and/or increased half-life as compared to an IgG comprising the lightchain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ IDNo.12.

Throughout the specification the term “human IgG1 constant domain”encompasses all allotypes and variants thereof known to a person skilledin the art.

In one aspect, the invention relates to an antigen binding protein whichspecifically binds to TNF-alpha comprising CDRH1 (SEQ ID NO: 27), CDRH2(SEQ ID NO: 28), CDRH3 (SEQ ID No: 29), CDRL1 (SEQ ID NO: 30), CDRL2(SEQ ID NO: 31), and CDRL3 (SEQ ID NO: 32); or variants thereof whereinsaid variants may contain 1, 2, 3 or 4 amino acid substitutions,insertions or deletions as compared to CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, or CDRL3; and a neonatal Fc receptor (FcRn) binding portion of ahuman IgG1 constant domain comprising one of more amino acidsubstitutions relative to the human IgG1 constant domain, wherein theantigen binding protein has an increased half life as compared to an IgGcomprising the light chain sequence of SEQ ID No. 2 and heavy chainsequence of SEQ ID No.12 and the antigen binding protein can beadministered no more than once every four weeks to achieve comparablemean steady-state trough concentration as that achieved by the same doseof IgG comprising the light chain sequence of SEQ ID No. 2 and the heavychain sequence of SEQ ID No.12 administered once every two weeks.

In one aspect, the invention relates to an antigen binding protein whichspecifically binds to TNF-alpha comprising CDRH1 (SEQ ID NO: 27), CDRH2(SEQ ID NO: 28), CDRH3 (SEQ ID No: 29), CDRL1 (SEQ ID NO: 30), CDRL2(SEQ ID NO: 31), and CDRL3 (SEQ ID NO: 32) or variants thereof whereinsaid variants may contain 1, 2, 3 or 4 amino acid substitutions,insertions or deletions as compared to CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, or CDRL3; and an FcRn binding portion of a human IgG1 constantdomain comprising one of more amino acid substitutions relative to thehuman IgG1 constant domain, wherein the antigen binding protein has anaffinity for FcRn of 2 fold, or 3 fold, or 4 fold or 5 fold, or 6 foldor 8 fold or greater than an anti-TNF antigen binding protein with thesame CDR's without such modifications at pH 6 as assessed by PrateOnXPR36 protein interaction array system at 25° C., the array systemhaving antigen binding proteins immobilised on the chip.

In one aspect, the invention relates to an antigen binding protein whichis a variant of an IgG comprising the light chain sequence of SEQ ID No.2 and the heavy chain sequence of SEQ ID No.12, wherein the antigenbinding protein variant comprises one or more substitutions in theneonatal Fc receptor (FcRn) binding portion of the IgG constant domainto increase the half-life of the antigen binding protein variantcompared with the IgG without such substitutions, wherein when thevariant is administered to patients at a single dose of 40 mg at a fourto eight weekly interval, the mean steady-state trough concentration inthe patient population does not fall below 4 μg/ml or does not fallbelow 5 μg/ml between dosing intervals. Preferably, the mean serumtrough antibody concentration in the patient population does not fallbelow 6 μg/ml between dosing intervals. Preferably, the mean serumtrough antibody concentration in the patient population does not fallbelow 5 μg/ml between dosing intervals when the variant is administeredto patients at a single dose of 40 mg at an eight weekly interval.Preferably, the mean serum trough antibody concentration in the patientpopulation does not fall below 4 μg/ml between dosing intervals whilststill providing the optimal efficacy when the variant is administered topatients at a single dose of 40 mg at an eight weekly interval.Preferably, the mean serum trough antibody concentration in the patientpopulation does not fall below 3 μg/ml between dosing intervals whilststill providing the optimal efficacy when the variant is administered topatients at a single dose of 40 mg at an eight weekly interval.

In one aspect, the invention relates to an antigen binding protein asdisclosed herein for treatment of a disease wherein the antigen bindingprotein can be administered to patients no more than once every fourweeks to achieve comparable mean steady-state trough concentration asthat achieved by the same dose of an IgG comprising light chain sequenceof SEQ ID No. 2 and heavy chain sequence of SEQ ID No.12 administeredonce every two weeks.

In one aspect, the invention relates to a method of treating a patientwith a disease, the method comprising administering an antigen bindingprotein according to the invention.

In one aspect, the invention relates to a nucleic acid sequence encodingthe antigen binding protein according to the invention, or a partthereof such as a heavy or light chain. In one aspect, the inventionrelates to an expression vector encoding the antigen binding proteinaccording to the invention, or a part thereof such as a heavy or lightchain.

In one aspect, the invention relates to a host cell comprising thenucleic acid sequence encoding the antigen binding protein according tothe invention. In one aspect, the invention relates to an antigenbinding protein according to the invention for use in the treatment ofPsoriasis or rheumatoid arthritis.

In one aspect, the invention relates to a kit comprising the antigenbinding protein according to the invention, and optionally comprisingmethotrexate for concomitant delivery of antigen binding proteinaccording to the invention and methotrexate.

In one aspect, the invention relates to an antigen binding protein asdisclosed herein for treatment of Rheumatoid arthritis in an individualwho is already being treated with methotrexate, and to an antigenbinding protein in combination with methotrexate for treatment ofRheumatoid arthritis, wherein the combination is deliveredsimultaneously, substantially simultaneously, or sequentially.

In one aspect, the invention relates to an antigen binding protein asdisclosed herein for treatment of Psoriasis in an individual who isalready being treated with methotrexate, and to an antigen bindingprotein in combination with methotrexate for treatment of Psoriasis,wherein the combination is delivered simultaneously, substantiallysimultaneously, or sequentially.

BRIEF DESCRIPTION OF FIGURES

FIG. 1—Binding of anti-TNFα antibodies to human TNFα

FIG. 2—Analysis of binding activity of anti-TNFα antibodies to humanTNFα following an accelerated stressor study

FIG. 3—Binding of anti-TNFα antibodies to human TNFα followingincubation in 25% human serum for 2 weeks

FIG. 4—Binding of anti-TNFα antibodies to human TNFα followingfreeze-thaw

FIG. 5—Analysis of anti-TNFα antibodies to FcγRIIIa receptors (a)Binding to human FcγRIIIa (valine 158 variant) (b) Binding to humanFcγRIIIA (phenylalanine 158 variant)

FIG. 6—Average dose normalised plasma concentrations of BPC2604 infemale cynomolgus monkeys and pascolizumab in male cynomolgus monkeysfollowing a single intravenous (1 hr infusion)

DETAILED DESCRIPTION OF INVENTION

The invention relates to novel antigen binding proteins bindingspecifically to TNF-alpha. In particular, the invention relates to novelvariants of anti-TNF antibodies such as adalimumab which show increasedbinding to the FcRn receptor and/or increased half life as compared toadalimumab. Adalimumab is an IgG monoclonal antibody comprising thelight chain sequence of SEQ ID No. 2 and heavy chain sequence of SEQ IDNo.12.

The inventors have found that specific modifications to adalimumab asdescribed herein show particular improvements in FcRn binding as shownin the examples below. Affinity matured variants of adalimumab also showimprovement in anti-TNF-alpha binding and/or neutralisation activity.

The novel antigen binding proteins of the invention have an increasedbinding to the FcRn receptor and/or increased half life and/or increasedMean Residence Time and/or decreased Clearance. It is considered thatbinding to FcRn results in longer serum retention in vivo. In order toincrease the retention of the Fc proteins in vivo, the increase inbinding affinity is observed around pH 6. In one aspect, the presentinvention therefore provides an antigen binding protein with optimisedbinding to FcRn.

In one embodiment, the half-life of the antigen binding protein of thepresent invention is increased 2 to 6 fold, such as 2 fold, 3 fold, 4fold, 5 fold or 6 fold as compared to an IgG comprising the light chainsequence of SEQ ID No. 2 and heavy chain sequence of SEQ ID No.12.Preferably, the half-life of the antigen binding protein of theinvention is increased 3 fold, 4 fold, or more compared to an IgGcomprising the light chain sequence of SEQ ID No. 2 and heavy chainsequence of SEQ ID No.12. For example, if the IgG is adalimumab having ahalf life of 10 days or in the range of 10 to 20 days then in oneembodiment an antigen binding protein of the present invention shows ahalf life of about 40 to 80 days. For example an antigen binding proteincomprising a heavy chain sequence selected from SEQ ID NO:5 or SEQ IDNO:9 or SEQ ID NO:15 or SEQ ID NO:18. or SEQ ID NO:21. or SEQ ID NO:24or SEQ ID NO:163, or SEQ ID NO:165, or SEQ ID NO:167, or SEQ ID NO:169.

In one embodiment, the antigen binding protein of the inventionadministered no more than once every four weeks in patients, achievesmean steady-state trough concentrations between about 2 μg/ml to about 7μg/ml. Preferably, the mean steady-state trough concentrations arebetween about 4 μg/ml to about 7 μg/ml and more preferably between about5 μg/ml to about 6 μg/ml.

In one embodiment, the antigen binding protein of the inventionadministered no more than once every 28 days in patients, achieves meansteady-state trough concentrations between about 2 μg/ml to about 7μg/ml. Preferably, the mean steady-state trough concentrations arebetween about 4 μg/ml to about 7 μg/ml and more preferably between about5 μg/ml to about 6 μg/ml.

In one embodiment of the invention, the antigen binding protein of theinvention can be administered once every 4, 5, 6, 7 or 8 weeks toachieve comparable mean steady-state trough concentrations as thoseachieved by adalimumab, when administered once every two weeks at thesame dose.

In a preferred embodiment of all aspects of the invention, the antigenbinding protein of the invention can be administered once every 7 or 8weeks.

In one embodiment of the invention, the antigen binding protein of theinvention can be administered once every 25-80 days for example onceevery 40-60 days, or for example once every 28, 35, 42, 49 or 56 days toachieve comparable mean steady-state trough concentrations as thoseachieved by adalimumab, when administered once every 14 days at the samedose.

In one embodiment of the invention, the antigen binding protein can beadministered once every 49 to 60 day, for example every 56 days.

In an embodiment of all aspects of the invention, the antigen bindingprotein has a 2 fold, or 4 fold, or 6 fold, or 8 fold or greateraffinity for human FcRn at pH 6 as assessed by PrateOn XPR36 proteininteraction array system at 25° C. wherein the antibodies areimmobilised on the chip. Preferably, the antigen binding protein has anaffinity for human FcRn between about 100 to about 500 KD (nM), such asbetween about 130 to about 360 KD (nM) or between about 140 to about 250KD (nM) or between about 140 to about 210 KD (nM).

In one embodiment, the clearance of the antigen binding protein is about2 to about 10 ml/hr, preferably about 2 to about 5 ml/hr or 2 to 4 ml/hror 2 to 3 ml/hr, such as about 2, about 2.5, 3, 4 or 5 ml/hr. In oneembodiment the antigen binding protein of the invention shows aclearance rate which is 2 fold, 3 fold, 4 fold or 5 fold lower thanadalimumab. In one embodiment, clearance for an antigen binding proteinaccording to the invention is in the ranges specified above or 2 fold, 3fold, 4 fold or 5 fold lower than adalimumab at a human dose of about 40mg.

In one aspect, the antigen binding protein of the invention is a variantof adalimumab (IgG comprising the light chain sequence of SEQ ID No. 2and the heavy chain sequence of SEQ ID No.12), the variant comprisingone or more substitutions in the FcRn binding portion of the IgGconstant domain to increase the half-life of the variant compared withadalimumab, wherein when the variant is administered to patients at asingle dose of 40 mg at a four to eight weekly interval, preferablyeight weekly interval, the mean steady-state trough antibodyconcentration in the patient population does not fall below 5 μg/ml. Inone embodiment the mean steady-state trough antibody concentration inthe patient population does not fall below 6 μg/ml, between dosingintervals.

In a further embodiment, the antigen binding protein comprises at leastone amino acid modification in the Fc region of said antigen bindingprotein, wherein said modification is at one or more of positions 250,252, 254, 256, 257, 259, 308, 428 or 434 of the Fc region as compared tosame position in the adalimumab sequence, wherein the numbering of theamino acids in the Fc region is that of the EU index in Kabat.

The wild type human IgG1 has amino acid residuesVal-Leu-His-Gln-Asp-Trp-Leu at positions 308-314, amino acid residuesLeu-Met-Ile-Ser-Arg-Thr at positions 251-256, amino acid residuesMet-His-Glu-Ala-Leu-His-Asn-HisTyr at positions 428-436, and amino acidresidues Gly-Gln-Pro-Glu-Asn at positions 385-389. Residue numbering maydiffer for IgG2-4.

In one embodiment, the antigen binding protein of the inventioncomprises one or more amino acid substitution relative to the human IgG1constant domain comprising the sequence of SEQ ID No. 13.

In one embodiment, the one or more amino acid substitution in the FcRnbinding portion of the human IgG1 heavy chain constant domain is atamino acid residues 252, 254 and 256 numbered according to EU index ofKabat and the aa substitution at residue 252 is a substitution of metwith tyr, phe, tryp or thr; the aa substitution at residue 254 is asubstitution of ser with thr; and the aa substitution at residue 256 isa substitution of thr with ser, arg, glu, asp or thr. Preferably, the aasubstitution at residue 252 is a substitution with tyr; the aasubstitution at residue 254 is a substitution with thr and thesubstitution at residue 256 is a substitution with glu. Preferably, theIgG1 constant domain is as shown in SEQ ID No: 7.

In one embodiment, the one or more amino acid substitutions in the FcRnbinding portion of the human IgG1 constant domain is at amino acidresidues 250 and 428 numbered according to EU index of Kabat and the aasubstitution at residue 250 is a substitution of thr with glu or gln;the aa substitution at residue 428 is a substitution of met with leu orphe. Preferably, the aa substitution at residue 250 is a substitutionwith glu and the aa substitution at residue 428 is a substitution withleu. Preferably, the IgG1 constant domain is as shown in SEQ ID No: 16.

In one embodiment, the one or more amino acid substitution in the FcRnbinding portion of the human IgG1 constant domain is at amino acidresidues 428 and/or 434 numbered according to EU index of Kabat.Preferably, the aa substitution at residue 428 is a substitution of metwith leu and the aa substitution at residue 434 is a substitution of asnwith ser. Preferably, the IgG1 constant domain is as shown in SEQ ID No:10.

In one embodiment, the one or more amino acid substitution in the FcRnbinding portion of the human IgG1 constant domain is at amino acidresidues 259 or 308 numbered according to EU index of Kabat. Preferably,the substitution at residue 259 is a substitution of val with ile andthe aa substitution at residue 308 is a substitution of val with phe.Preferably, the IgG1 constant domain is as shown in SEQ ID No: 19 or SEQID No: 22.

In one embodiment, the one or more amino acid substitution in the FcRnbinding portion of the human IgG1 heavy chain constant domain is atamino acid residues 257 and 434 numbered according to EU index of Kabatas shown in SEQ ID No: 25.

In one embodiment, the one or more amino acid substitution in the FcRnbinding portion of the human IgG1 heavy chain constant domain is atamino acid residues 433 and 434 numbered according to EU index of Kabatfor example the residues are H433K and N434F Preferably, the IgG1constant domain is as shown in SEQ ID No: 165 or SEQ ID No: 167.

In one embodiment, the antigen binding protein comprises any of the IgG1constant domain modifications listed in Table A.

In one embodiment, the antigen binding protein is an antibody.

In one embodiment, the antigen binding protein comprises a variabledomain of SEQ ID NO: 6 and/or SEQ ID NO: 3 or a variant thereof whichcontains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions,insertions or deletions and/or shares at least 90% identity across thelength of SEQ ID NO: 6 or SEQ ID NO: 3.

In one embodiment, the antigen binding protein comprises the heavy chainsequence as shown in SEQ ID No 5, 9 or 15 optionally with a light chainsequence as shown in SEQ ID No: 2.

In one embodiment, the antigen binding protein comprises a variableheavy domain sequence as shown in SEQ ID NO: 78 or 80.

In one embodiment, the antigen binding protein comprises a heavy chainsequence as shown in SEQ ID NO: 145 or SEQ ID NO: 146 optionally with alight chain variant as shown in SEQ ID Nos. 148, 150 or 152.

In one embodiment, the antigen binding protein comprises the heavy chainsequence as shown in SEQ ID No 18 or 21 optionally with a light chainsequence as shown in SEQ ID No: 2.

In one embodiment the antigen binding protein according to the inventioncomprises any of the variable domains specified in Table A. In oneembodiment, the antigen binding protein according to the inventioncomprises the variable heavy domain having the sequence of cb1-3-VH,cb2-44-VH, cb1-39-VH, cb1-31-VH, cb2-11-VH, cb2-40-VH, cb2-35-VH,cb2-28-VH, cb2-38-VH, cb2-20-VH, cb1-8-VL or cb1-43-VL as shown in TableA.

In one embodiment, the antigen binding protein according to theinvention comprises the variable light domain having the sequence ofcb1-45-VL, cb1-4-VL, cb1-41-VL, cb1-37-VL, cb1-39-VL, cb1-33-VL,cb1-35-VL, cb1-31-VL, cb1-29-VL, cb1-22-VL, cb1-23-VL, cb1-12-VL,cb1-10-VL, cb2-1-VL, cb2-11-VL, cb2-40-VL, cb2-35-VL, cb2-28-VL,cb2-20-VL, cb1-3-VL, cb2-6-VL or cb2-44-VL as shown in Table A.

For example, the antigen binding protein according to the inventioncomprises a variable domain having the sequence of cb1-3VH, cb2-44VH orcb2-6VL as shown in Table A.

In one embodiment the antigen binding protein according to the inventioncomprises any of the variable domains specified in Table A. In oneembodiment, the antigen binding protein according to the inventioncomprises the variable heavy domain having a sequence selected from SEQID NO: 170 or SEQ ID NO: 174 or SEQ ID NO:178

In one embodiment, the antigen binding protein according to theinvention comprises the variable light domain having a sequence selectedfrom SEQ ID NO: 171 or SEQ ID NO: 175 or SEQ ID NO:179

In a further embodiment the antigen binding protein comprises any of theIgG1 constant domain modifications listed in Table A.

Variants of all the above mentioned variable domains or heavy chainsequences or light chain sequences which contain 1, 2, 3, 4, 5, 6, 7, 8,9 or 10 amino acid substitutions, insertions or deletions and/or shareat least 90% identity across the length of any of these sequences arealso within the scope of the invention.

In one embodiment, the antigen binding protein of the inventioncomprises a variant of CDRH3 (SEQ ID No: 29) which variant has 1, 2, 3or 4 amino acid substitutions as compared to SEQ ID No: 29. In oneembodiment, the variant CDRH3 may have the sequence as shown in any oneof SEQ ID Nos. 40 to 49.

In one embodiment, the antigen binding protein of the inventioncomprises a variant of CDRH1 (SEQ ID No: 27) which variant has 1 or 2amino acid substitutions as compared to SEQ ID No: 27. In oneembodiment, the variant CDRH1 may have the sequence as shown in any oneof SEQ ID Nos. 33 to 38.

In one embodiment, the antigen binding protein of the inventioncomprises a variant of CDRL1 (SEQ ID No: 30) which variant has 1, 2 or 3amino acid substitutions as compared to SEQ ID No: 30. In oneembodiment, the variant CDRL1 may have the sequence as shown in any oneof SEQ ID Nos. 50 to 61.

In one embodiment, the antigen binding protein of the inventioncomprises a variant of CDRL2 (SEQ ID No: 31) which variant has 1, 2 or 3amino acid substitutions as compared to SEQ ID No: 31. In oneembodiment, the variant CDRL2 may have the sequence as shown in any oneof SEQ ID Nos. 62 to 72.

In one embodiment, the antigen binding protein of the inventioncomprises a variant of CDRL3 (SEQ ID No: 32) which variant has 1, 2 or 3amino acid substitutions as compared to SEQ ID No: 32. In oneembodiment, the variant CDRL3 may have the sequence as shown in any oneof SEQ ID Nos. 73 to 76.

In one embodiment, the invention relates to an antigen binding proteinwhich specifically binds to TNF-alpha comprising one or more or all CDRsselected from: CDRH1 (SEQ ID NO: 27), CDRH2 (SEQ ID NO: 28), CDRH3 (SEQID No: 29), CDRL1 (SEQ ID NO: 30), CDRL2 (SEQ ID NO: 31), and CDRL3 (SEQID NO: 32); wherein any of the CDRs could be a variant CDR whichcontains 1, 2, 3 or 4 amino acid substitutions, insertions or deletionsas compared to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3. In oneaspect, the antigen binding protein of the invention comprises CDRH1,CDRH3, CDRL1, CDRL2 and CDRL3 wherein any of the CDRs could be a variantCDR which contains 1, 2, 3 or 4 amino acid substitutions, insertions ordeletions compared to CDRH1, CDRH3, CDRL1, CDRL2, or CDRL3. In oneaspect, the antigen binding protein of the invention comprises CDRH1,CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 wherein any of the CDRs could be avariant CDR which contains 1, 2, 3 or 4 amino acid substitutions,insertions or deletions compared to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2,or CDRL3

In one aspect, the invention relates to a method of treating a humanpatient with a disease, the method comprising administering an antigenbinding protein according to the invention.

The invention also relates to an antigen binding protein as disclosedherein for the treatment of disease in a human.

The invention also relates to use of an antigen binding protein asdisclosed herein in the manufacture of a medicament for the treatment ofdisease, and an antigen binding protein as disclosed herein for use intreatment of disease.

In one embodiment, the disease to be treated by the antigen bindingprotein of the invention is rheumatoid arthritis, polyarticular juvenileidiopathic arthritis, psoriatic arthritis, ankylosing spondylitis,Ulcerative colitis, spondyloarthropathy, Crohn's disease or Psoriasis.

In one embodiment, the antigen binding protein of the invention is to beadministered with methotrexate. The methotrexate can be deliveredbefore, after or at the same time, or substantially the same time, asthe antigen binding protein. In a preferred embodiment the antigenbinding protein of the invention is to be administered with methotrexateto a patient suffering from rheumatoid arthritis. In one embodiment,methotrexate is administered to patients receiving an antigen bindingprotein of the invention to reduce the immunogenic effect of the antigenbinding protein. In one embodiment, the antigen binding protein of theinvention is administered to patients already receiving methotrexate.Methotrexate may be substituted by another acceptable compound whichreduced the immune response to the antigen binding protein, for examplecorticosteroids.

In one aspect, the invention relates to a method of treating a patientwith a disease, the method comprising administering an antigen bindingprotein of the invention. In one embodiment, the method comprisesadministering an antigen binding protein to the patient as a single 20,30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 mg dose no more than onceevery four weeks, preferably once every 5, 6, 7, or 8 weeks and mostpreferably once every 8 weeks. Preferably, the dose is 40 to 80 mg, forexample 40 mg.

The invention also provides a polynucleotide sequence encoding any aminoacid sequence disclosed herein, including a heavy chain of any of theantigen binding constructs described herein, and a polynucleotideencoding a light chain of any of the antigen binding constructsdescribed herein. Such polynucleotides represent the coding sequencewhich corresponds to the equivalent polypeptide sequences, however itwill be understood that such polynucleotide sequences could be clonedinto an expression vector along with a start codon, an appropriatesignal sequence and a stop codon. The polynucleotide may be DNA or RNA.

The invention also provides a host cell, for example a recombinant,transformed or transfected cell, comprising one or more polynucleotidesencoding a heavy chain and/or a light chain of any of the antigenbinding constructs described herein.

The invention further provides a pharmaceutical composition comprisingan antigen binding construct as described herein a pharmaceuticallyacceptable carrier.

The invention further provides a method for the production of any of theantigen binding constructs described herein which method comprises thestep of culturing a host cell comprising a first and second vector, saidfirst vector comprising a polynucleotide encoding a heavy chain of anyof the antigen binding constructs described herein and said secondvector comprising a polynucleotide encoding a light chain of any of theantigen binding constructs described herein, in a serum-free/chemicallydefined/animal derived component free culture media. Alternatively amethod may comprise culturing a host cell comprising a vector comprisinga polynucleotide encoding a heavy chain of any of the antigen bindingconstructs described herein and a polynucleotide encoding a light chainof any of the antigen binding constructs described herein, suitably in aserum-free/chemically defined/animal derived component free culturemedia.

In another embodiment, the invention includes a method of increasing thehalf-life of an antibody by modifying an Fc according to themodifications described herein.

In another embodiment, the invention includes an antigen binding proteinas described herein with enhanced FcRn binding and having one or moreadditional substitutions, deletions or insertions that modulate anotherproperty of the effector function.

Once expressed by the desired method, the antigen binding protein of theinvention is then examined for in vitro activity by use of anappropriate assay. Presently conventional ELISA and Biacore assayformats are employed to assess qualitative and quantitative binding ofthe antigen binding construct to its target. Additionally, other invitro assays may also be used to verify neutralizing efficacy prior tosubsequent human clinical studies performed to evaluate the persistenceof the antigen binding protein in the body despite the usual clearancemechanisms.

The dose and duration of treatment relates to the relative duration ofthe molecules of the present invention in the human circulation, and canbe adjusted by one of skill in the art depending upon the conditionbeing treated and the general health of the patient based on theinformation provided herein. It is envisaged that repeated dosing (e.g.once every 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks) over anextended time period (e.g. four to six months) maybe required to achievemaximal therapeutic efficacy.

The mode of administration of the therapeutic agent of the invention maybe any suitable route which delivers the agent to the host. The antigenbinding proteins, and pharmaceutical compositions of the invention areparticularly useful for parenteral administration, i.e., subcutaneously(s.c.), intrathecally, intraperitoneally, intramuscularly (i.m.),intravenously (i.v.), or intranasally. In one embodiment the antigenbinding proteins and pharmaceutical compositions of the invention areadministered via a subcutaneous auto injector pen or a subcutaneouspre-filled syringe.

Antigen binding proteins of the invention may be prepared aspharmaceutical compositions containing an effective amount of theantigen binding protein of the invention as an active ingredient in apharmaceutically acceptable carrier. In the prophylactic agent of theinvention, an aqueous suspension or solution containing the antigenbinding construct, preferably buffered at physiological pH, in a formready for injection is preferred. The compositions for parenteraladministration will commonly comprise a solution of the antigen bindingconstruct of the invention or a cocktail thereof dissolved in apharmaceutically acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be employed, e.g., 0.9% saline, 0.3%glycine, and the like. These solutions may be made sterile and generallyfree of particulate matter. These solutions may be sterilized byconventional, well known sterilization techniques (e.g., filtration).The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, etc. The concentration of the antigenbinding protein of the invention in such pharmaceutical formulation canvary widely, i.e., from less than about 0.5%, usually at or at leastabout 1% to as much as 15 or 20% by weight and will be selectedprimarily based on fluid volumes, viscosities, etc., according to theparticular mode of administration selected.

It has been reported that adalimumab is difficult to formulate at highconcentrations. WO2004016286 describes an adalimumab formulationcomprising a citrate-phosphate buffer and other components including apolyol and a detergent. The oral presentation “Humira®—from Developmentto Commercial Scale Production” presented on 25 Oct. 2005 at the PDAConference reports formulations comprising (i) citrate-phosphate buffer;(ii) acetate-phosphate buffer; and (iii) phosphate buffer. Theacetate-phosphate buffer tested displayed the worst stabilising effectupon adalimumab. Curtis et al. (2008) Current Medical Research andOpinion, Volume 27, p 71-78, report the incidence of injection-siteburning and stinging in patients with rheumatoid arthritis usinginjectable adalimumab. The burning and stinging has been partlyattributed to citrate buffer-based formulations (Basic and ClinicalPharmacology & Toxicology, Volume 98, p 218-221, 2006; and Journal ofPharmaceutical Sciences, Volume 97, p 3051-3066, 2008). However,WO20100129469 describes a high adalimumab concentration formulation thatstill comprises a citrate-phosphate buffer and other componentsincluding a polyol with no sodium chloride. The more recent WO2012065072describes an adalimumab formulation comprising a surfactant and a polyolwith no buffer, thus potentially avoiding any citrate buffer effectsupon injection.

In one embodiment there is provided a liquid formulation comprising aTNF-alpha antigen binding protein and an acetate buffer. In a furtherembodiment the TNF-alpha binding protein comprises a CDRH1 selected fromSEQ ID NO:27 or SEQ ID NO:'s 33-38 and/or a CDRH2 of SEQ ID NO:28 and/ora CDRH3 selected from SEQ ID NO:29 or SEQ ID NO:'s 40-49 and/or a CDRL1selected from SEQ ID NO:30 or SEQ ID NO:'s 50-61 and/or a CDRL2 selectedfrom SEQ ID NO:31 or SEQ ID NO:'s 62-72 and/or a CDRL3 of SEQ ID NO:32or SEQ ID NO:'s73-76. For example the TNF-alpha antigen binding proteincomprises CDRH1 of SEQ ID NO:27 and CDRH2 of SEQ ID NO:28 and CDRH3 ofSEQ ID NO:29 and CDRL1 of SEQ ID NO:30 and CDRL2 selected from SEQ IDNO:31 and a CDRL3 of SEQ ID NO:32 or variants thereof.

The TNF-alpha antigen binding protein may be adalimumab. The TNF-alphaantigen binding protein may be BPC1494. The TNF-alpha antigen bindingprotein may be BPC 1496.

The TNF-alpha antigen binding proteins described herein are formulatedin an acetate buffer. The formulation may be in liquid form. Theformulation may further comprise one or more, a combination, or all of:a surfactant; a chelator; a salt; and an amino acid. The TNF-alphaantigen binding proteins are formulated at high concentrations, forexample at 50 mg/mL. In one embodiment, the formulation does notcomprise a polyol. In another embodiment, the formulation does notcomprise a further buffer component, for example citrate. Therefore, theformulations described herein solve the problem of providing TNF-alphaantigen binding proteins, in particular the TNF-alpha antigen bindingproteins as described in Table A, at high concentrations in a stableformulation, and avoid the burning and stinging effects of citrate-basedbuffers.

In one embodiment, the acetate buffer formulation further comprises asurfactant and a chelator. In another embodiment, the acetate bufferformulation further comprises a surfactant and a salt. In anotherembodiment, the acetate buffer formulation further comprises asurfactant and an amino acid. In another embodiment, the acetate bufferformulation further comprises a chelator and a salt. In anotherembodiment, the acetate buffer formulation further comprises a chelatorand an amino acid. In another embodiment, the acetate buffer formulationfurther comprises a salt and an amino acid.

In one embodiment, the acetate buffer formulation further comprises asurfactant, a chelator, and a salt. In another embodiment, the acetatebuffer formulation further comprises a surfactant, a chelator, and anamino acid. In another embodiment, the acetate buffer formulationfurther comprises a surfactant, a salt, and an amino acid. In anotherembodiment, the acetate buffer formulation further comprises a chelator,a salt, and an amino acid.

In one embodiment, the buffer is sodium acetate trihydrate. This may beat a concentration of 10 to 100 mM sodium acetate trihydrate (1.361 to13.61 mg/mL). Sodium acetate trihydrate may be present in an amount of20 to 80 mM, 30 to 70 mM, 40 to 60 mM, or about 40 mM, about 45 mM,about 50 mM, about 55 mM, or about 60 mM. In one embodiment, sodiumacetate trihydrate is at a concentration of about 50 mM (6.80 mg/mL).

The acetate buffer may be the sole buffer. In other words, theformulation may not comprise another buffer component, such as phosphateor citrate buffer. Citrate buffer may be detrimental to the formulationfor a number of reasons: (i) it may not be a good buffer because thevalues of the three dissociation constants are too close to permitdistinction of the three proton receptor phases; (ii) citrate may act asa metal chelator and thus influence metal ion balance: (iii) citrate isa metabolite of the citric acid cycle and has the potential to influencecellular metabolism.

Suitable surfactants (also known as detergents) may include, e.g.,polysorbates (for example, polysorbate 20 or 80), polyoxyethylene alkylethers such as Brij 35®, poloxamers (for example poloxamer 188,Poloxamer 407), Tween 20, Tween 80, Cremophor A25, Sympatens ALM/230,and Mirj. In one embodiment, the surfactant is polysorbate 80. Theformulation may comprise a concentration of 0.01 to 0.1% polysorbate 80(0.1 to 1 mg/mL). Polysorbate 80 may be present in an amount of 0.01 to0.05%, or 0.01 to 0.03%; or about 0.015%, about 0.02%, or about 0.025%.In one embodiment, polysorbate 80 is at a concentration of about 0.02%w/v (0.2 mg/mL). A high concentration of polysorbate 80, for examplemore than 0.1%, may be detrimental to the formulation because thissurfactant may contain high levels of oxidants which may increase levelsof oxidation upon storage of the formulation and therefore reduce shelflife.

Suitable chelating agents may include EDTA and metal complexes (e.g.Zn-protein complexes). In one embodiment, the chelating agents is EDTA.The formulation may comprise a concentration of 0.02 to 0.2 mM EDTA(0.00748 to 0.0748 mg/mL). EDTA may be present in an amount of 0.02 to0.15 mM, 0.02 to 0.1 mM, 0.03 to 0.08 mM, or 0.04 to 0.06 mM; or about0.03 mM, about 0.04 mM, about 0.05 mM, or about 0.06 mM. In oneembodiment, EDTA is at a concentration of about 0.05 mM (0.018 mg/mL).

Suitable salts may include any salt-forming counterions, such as sodium.For example, sodium chloride may be used, or anionic acetate instead ofchloride as a counterion in a sodium salt may be used. In oneembodiment, the salt is sodium chloride. The formulation may comprise aconcentration of 25 to 100 mM sodium chloride (1.461 to 5.84 mg/mL).Sodium chloride may be present in an amount of 35 to 90 mM, 45 to 80 mM,25 to 70 mM, or 45 to 60 mM; or 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM. In one embodiment, sodiumchloride is at a concentration of about 51 mM (2.98 mg/mL).

Suitable amino acids may include arginine. The formulation may comprisea concentration of 0.5 to 5% arginine free base (5 to 50 mg/mL).Arginine free base may be present in an amount of In other embodiments,the arginine free base may be between 0.5 to 4.0%, 0.5 to 3.5%, 0.5 to3.0%, 0.5 to 2.5%, or about 0.5%, about 0.75%, about 1%, about 1.5%,about 2%, or about 3%. In one embodiment, arginine is at a concentrationof about 1% (10 mg/mL).

A polyol is a substance with multiple hydroxyl groups, and includessugars (reducing and non-reducing sugars), sugar alcohols and sugaracids. Examples of polyols include fructose, mannose, maltose, lactose,arabinose, xylose, ribose, rhamnose, galactose, glucose, sucrose,trehalose, sorbose, melezitose, raffinose, mannitol, xylitol,erythritol, threitol, sorbitol, glycerol, L-gluconate and metallic saltsthereof. In one embodiment, the formulation of the invention does notcomprise a polyol.

In one embodiment, the acetate buffer formulation further comprises oneor more, a combination, or all of: polysorbate 80, EDTA, sodiumchloride, and arginine free base.

The pH of the formulation may be adjusted to pH 5.0 to 7.0. In oneembodiment, acetic acid is present (about 100 mM acetic acid) to adjustthe formulation to about pH 5.5. In other embodiments, the pH may beadjusted to pH 5.0, 5.5, 6.0, 6.5 or 7.0. In yet other embodiments ofthe invention, NaOH or HCl is used to adjust the pH to 5.0, 5.5, 6.0,6.5 or 7.0.

The TNF-alpha antigen binding proteins described herein may beformulated in the concentration range of 20 to 300 mg/mL. For example,the antigen binding protein is present in a concentration of 20-200mg/mL or 50-100 mg/mL; or about 40 mg/mL or about 45 mg/mL or about 50mg/mL or about 55 mg/mL or about 60 mg/mL or about 70 mg/mL or about 80mg/mL or about 90 mg/mL, or about 100 mg/mL. In one embodiment, theTNF-alpha antigen binding protein is at a concentration of about 50mg/mL.

The TNF-alpha antigen binding protein may be adalimumab. The TNF-alphaantigen binding protein may be BPC1494. The TNF-alpha antigen bindingprotein may be BPC 1496.

In one embodiment, the formulation is stable for at least 1 year, atleast 18 months, or at least 2 years. For example, the formulation isstable at a temperature of about 5° C. for at least 1 year, at least 18months, or at least 2 years. In another embodiment, the formulation isstable at room temperature (about 25° C.). For example, the formulationis stable at a temperature of about 25° C. for at least 14 weeks, atleast 2 weeks, at least 1 week, at least 6 days, at least 5 days, atleast 4 days, at least 3 days, at least 2 days or at least 1 day. Inanother embodiment, the formulation is stable at a temperature of about40° C. For example, the formulation is stable at a temperature of about40° C. for at least 9 weeks or at least 4 weeks.

As shown by Examples 25 and 26 below, the formulations are stable atroom temperature (about 25° C.). Therefore, there is minimal risk ofaggregates or low molecular weight fragments forming in pre-filleddevices for injection that may be left at room temperature for more thanthe recommended time. Aggregates are potentially immunogenic (see TheAAPS Journal 2006; 8 (3) Article 59 Themed Issue: Proceedings of the2005 AAPS Biotec Open Forum on Aggregation of Protein Therapeutics,Guest Editor—Steve Shire, Effects of Protein Aggregates: An ImmunologicPerspective) and low molecular weight fragments may illicit pre-existingautoantibodies (see J Immunol 2008; 181:3183-3192; Human Anti-IgG1 HingeAutoantibodies Reconstitute the Effector Functions of ProteolyticallyInactivated IgGs1).

The stability of a TNF-alpha antigen binding protein in a liquidformulation may be assessed by any one or a combination of: appearanceby visual observation, protein concentration (A280 nm), size exclusionchromatography (SEC), Capillary Iso-Electric Focussing (c-IEF), and by afunctional binding assay (ELISA). For example, the percentage ofmonomer, aggregate, or fragment, or combinations thereof, can be used todetermine stability. In one embodiment, a stable liquid formulation is aformulation having less than about 10%, or less than about 5% of theTNF-alpha antigen binding protein being present as aggregate in theformulation. The formulation may have a monomer content of at least 95%,or at least 96%, or at least 97%, or at least 98%, or at least 99%. Theformulation may have a monomer content of at least 95%, or at least 96%,or at least 97%, or at least 98%, or at least 99% at room temperature(about 25° C.) after about 2 weeks. The formulation may have a monomercontent of at least 95%, or at least 96%, or at least 97%, or at least98%, or at least 99% at room temperature (about 25° C.) after about 1week. The formulation may have a monomer content of at least 95%, or atleast 96%, or at least 97%, or at least 98%, or at least 99% at roomtemperature (about 25° C.) after about 1 day.

Thus, a pharmaceutical composition of the invention for injection couldbe prepared to contain 1 mL sterile buffered water, and between about 1mg to about 100 mg, e.g. about 30 mg to about 100 mg or more preferably,about 35 mg to about 80 mg, such as 40, 50, 80 or 90 mg of an antigenbinding construct of the invention. Actual methods for preparingparenterally administrable compositions are well known or will beapparent to those skilled in the art and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa. For the preparation of intravenouslyadministrable antigen binding construct formulations of the inventionsee Lasmar U and Parkins D “The formulation of Biopharmaceuticalproducts”, Pharma. Sci. Tech. today, page 129-137, Vol. 3 (3 Apr. 2000),Wang, W “Instability, stabilisation and formulation of liquid proteinpharmaceuticals”, Int. J. Pharm 185 (1999) 129-188, Stability of ProteinPharmaceuticals Part A and B ed Ahern T. J., Manning M. C., New York,N.Y.: Plenum Press (1992), Akers, M. J. “Excipient-Drug interactions inParenteral Formulations”, J. Pharm Sci 91 (2002) 2283-2300, Imamura, Ket al “Effects of types of sugar on stabilization of Protein in thedried state”, J Pharm Sci 92 (2003) 266-274, Izutsu, Kkojima, S.“Excipient crystallinity and its protein-structure-stabilizing effectduring freeze-drying”, J. Pharm. Pharmacol, 54 (2002) 1033-1039,Johnson, R, “Mannitol-sucrose mixtures-versatile formulations forprotein lyophilization”, J. Pharm. Sci, 91 (2002) 914-922.

Preferably, the antigen binding protein of the invention is provided oradministered at a dose of about 40 mg. Preferably the antigen bindingprotein is suitable for subcutaneous delivery and is deliveredsubcutaneously. Other dosing or administration routes may also be used,as disclosed herein.

In one emboduiment the antigen binding proteins according to any aspectof the invention shows increased Mean Residence Time as compared to anIgG comprising the light chain sequence of SEQ ID No. 2 and heavy chainsequence of SEQ ID No.12.

The binding ability of modified IgGs and molecules comprising an IgGconstant domain or FcRn binding portion thereof can be characterized byvarious in vitro assays. PCT publication WO 97/34631 by Ward disclosesvarious methods in detail. For example, in order to compare the abilityof the modified IgG or fragments thereof to bind to FcRn with that ofthe wild type IgG, the modified IgG or fragments thereof and the wildtype IgG can be radio-labeled and reacted with FcRn-expressing cells invitro. The radioactivity of the cell-bound fractions can be then countedand compared. The cells expressing FcRn to be used for this assay aremay be endothelial cell lines including mouse pulmonary capillaryendothelial cells (B10, D2.PCE) derived from lungs of B10.DBA/2 mice andSV40 transformed endothelial cells (SVEC) (Kim et al., J Immunol., 40:457-465, 1994) derived from C3H/HeJ mice. However, other types of cellswhich express sufficient number of FcRn, including mammalian cells whichexpress recombinant FcRn of a species of choice, can be also used.Alternatively, after counting the radioactivity of the bound fraction ofmodified IgG or that of unmodified IgG, the bound molecules can be thenextracted with the detergent, and the percent release per unit number ofcells can be calculated and compared.

Affinity of antigen binding proteins of the inventions for FcRn can bemeasured by surface plasmon resonance (SPR) measurement using, forexample, a BIAcore 2000 (BIAcore Inc.) as described previously (Popov etal., Mol. Immunol., 33: 493-502, 1996; Karlsson et al., J. Immunol.Methods, 145: 229-240, 1991, both of which are incorporated by referencein their entireties). In this method, FcRn molecules are coupled to aBIAcore sensor chip (e.g., CM5 chip by Pharmacia) and the binding ofmodified IgG to the immobilized FcRn is measured at a certain flow rateto obtain sensorgrams using BIA evaluation 2.1 software, based on whichon- and off-rates of the modified IgG, constant domains, or fragmentsthereof, to FcRn can be calculated. Relative affinities of antigenbinding proteins of the invention and unmodified IgG for FcRn can bealso measured by a simple competition binding assay. Furthermore,affinities of modified IgGs or fragments thereof, and the wild type IgGfor FcRn can be also measured by a saturation study and the Scatchardanalysis.

Transfer of modified IgG or fragments thereof across the cell by FcRncan be measured by in vitro transfer assay using radiolabeled IgG orfragments thereof and FcRn-expressing cells and comparing theradioactivity of the one side of the cell monolayer with that of theother side. Alternatively, such transfer can be measured in vivo byfeeding 10- to 14-day old suckling mice with radiolabeled, modified IgGand periodically counting the radioactivity in blood samples whichindicates the transfer of the IgG through the intestine to thecirculation (or any other target tissue, e.g., the lungs). To test thedose-dependent inhibition of the IgG transfer through the gut, a mixtureof radiolabeled and unlabeled IgG at certain ratio is given to the miceand the radioactivity of the plasma can be periodically measured (Kim etal., Eur. R Immunol., 24: 2429-2434, 1994).

The half-life of antigen binding proteins can be measured bypharmacokinetic studies according to the method described by Kim et al.(Eur. J. of Immuno. 24: 542, 1994), which is incorporated by referenceherein in its entirety. According to this method, radiolabeled antigenbinding protein is injected intravenously into mice and its plasmaconcentration is periodically measured as a function of time, forexample, at 3 minutes to 72 hours after the injection. The clearancecurve thus obtained should be biphasic. For the determination of the invivo half-life of the modified IgGs or fragments thereof, the clearancerate in β-phase is calculated and compared with that of the unmodifiedIgG.

Antigen binding proteins of the invention may be assayed for the abilityto immunospecifically bind to an antigen. Such an assay may be performedin solution (e.g., Houghten, BiolTechniques, 13: 412-421, 1992), onbeads (Lam, Nature, 354: 82-84, 1991, on chips (Fodor, Nature, 364:555-556, 1993), on bacteria (U.S. Pat. No. 5,223,409), on spores (U.S.Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull etal., Proc. Natl. Acad. Sci. USA, 89: 1865-1869, 1992) or on phage (Scottand Smith, Science, 249: 386-390, 1990; Devlin, Science, 249: 404-406,1990; Cwirla et al., Proc. Natl. Acad. Sci. USA, 87: 6378-6382, 1990;and Felici, J: Mol. Biol., 222: 301-310, 1991) (each of these referencesis incorporated herein in its entirety by reference). Antibodies thathave been identified to immunospecifically bind to an antigen or afragment thereof can then be assayed for their specificity affinity forthe antigen.

The antigen binding proteins of the invention may be assayed forimmunospecific binding to an antigen and cross-reactivity with otherantigens by any method known in the art. Immunoassays which can be usedto analyze immunospecific binding and cross-reactivity include, but arenot limited to, competitive and non-competitive assay systems usingtechniques such as western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of antibodies to an antigen. BIAcorekinetic analysis comprises analyzing the binding and dissociation of anantigen from chips with immobilized antibodies on their surface.

Antigen binding protein: The term “antigen binding protein” as usedherein includes reference to antibodies, antibody fragments and otherprotein constructs, which are capable of binding to TNF-alpha.

Antibody: The term “antibody” is used herein in the broadest sense andincludes reference to molecules with an immunoglobulin-like domain andincludes monoclonal, recombinant, polyclonal, chimeric, humanised,bispecific and heteroconjugate antibodies.

Human IgG1 heavy chain constant domain: refers to human amino acidsequence for the IgG1 heavy chain constant domain that is found innature, including allelic variations.

“Half-life (t½)” refers to the time required for the concentration ofthe antigen binding polypeptide to reach half of its original value. Theserum half-life of proteins can be measured by pharmacokinetic studiesaccording to the method described by Kim et al. (Eur. J. of Immuno. 24:542, 1994). According to this method, radiolabeled protein is injectedintravenously into mice and its plasma concentration is periodicallymeasured as a function of time, for example, at about 3 minutes to about72 hours after the injection. Other methods for pharmacokinetic analysisand determination of the half-life of a molecule will be familiar tothose skilled in the art. Details may be found in Kenneth, A et al:Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and inPeters et al, Pharmacokinetic analysis: A Practical Approach (1996).Reference is also made to “Pharmacokinetics”, M Gibaldi & D Perron,published by Marcel Dekker, 2nd Rev. ex edition (1982), which describespharmacokinetic parameters such as t alpha and t beta half lives andarea under the curve (AUC), and “Clinical Pharmacokinetics: Concepts andApplications”, Rowland and Tozer, Third Edition (1995).

“Clearance (CL)” refers to the volume of plasma irreversibly cleared ofa protein per unit time. Clearance is calculated as the Dose/AUC (AUC:is the Area Under Curve or Area under the plasma drug concentration timecurve). Clearance can also be calculated by the rate of drug eliminationdivided by the plasma concentration of the drug (rate ofelimination=CL*concentration)

“Mean Residence Time (MRT)” is the average time that the antigen bindingpolypeptides reside in the body before being irreversibly eliminated.Calculated as MRT=AUMC/AUC.

“Steady state concentration” (Css) is the concentration reached when thedrug elimination rate becomes equal to drug administration rate as aresult of continued drug administration. Css fluctuates between peak andtrough levels and is measured in microgram/ml. “Mean steady-state troughconcentration” refers to the mean of the trough level across the patientpopulation at a given time.

“Comparable mean steady-state trough concentration” refers to meansteady-state trough concentration which is the same or within about 10%to 30% of the stated value. Comparable mean steady-state troughconcentration for the antigen binding polypeptides of the invention maybe considered to be those mean steady-state trough concentrations thatare 0.8 to 1.25 times the mean steady-state trough concentrationachieved with an IgG comprising the light chain sequence of SEQ ID No. 2and the heavy chain sequence of SEQ ID No. 12.

Half lives and AUC can be determined from a curve of serum concentrationof drug (for example the antigen binding polypeptide of the presentinvention) against time. Half life may be determined throughcompartmental or non-compartmental analysis. The WINNONLIN™ analysispackage (available from Pharsight Corp., Mountain View, Calif. 94040,USA) can be used, for example, to model the curve. In one embodiment,“half life” refers to the terminal half life.

Specifically binds: The term “specifically binds” as used throughout thepresent specification in relation to antigen binding proteins means thatthe antigen binding protein binds to TNF-alpha with no or insignificantbinding to other unrelated proteins. The term however does not excludethe fact that the antigen binding proteins may also be cross-reactivewith closely related molecules. The antigen binding proteins describedherein may bind to TNF-alpha with at least 2, at least 5, at least 10,at least 50, at least 100, or at least 1000 fold greater affinity thanthey bind to closely related molecules.

CDRs:

“CDRs” are defined as the complementarity determining region amino acidsequences of an antigen binding protein. These are the hypervariableregions of immunoglobulin heavy and light chains. There are three heavychain and three light chain CDRs (or CDR regions) in the variableportion of an immunoglobulin. Thus, “CDRs” as used herein refers to allthree heavy chain CDRs, all three light chain CDRs, all heavy and lightchain CDRs, or at least two CDRs.

Throughout this specification, amino acid residues in variable domainsequences and full length antibody sequences are numbered according tothe Kabat numbering convention. Similarly, the terms “CDR”, “CDRL1”,“CDRL2”, “CDRL3”, “CDRH1”, “CDRH2”, “CDRH3” used in the Examples followthe Kabat numbering convention. For further information, see Kabat etal., Sequences of Proteins of Immunological Interest, 4th Ed., U.S.Department of Health and Human Services, National Institutes of Health(1987).

% identity of variants: The term “identical” or “sequence identity”indicates the degree of identity between two nucleic acid or two aminoacid sequences when optimally aligned and compared with appropriateinsertions or deletions. The variants described herein may have 90, 91,92, 93, 94, 95, 96, 97, 98, or 99% identity to the native CDR orvariable domain sequences at the amino acid level.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine study, numerous equivalents to the specific proceduresdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the claims. All publications andpatent applications mentioned in the specification are indicative of thelevel of skill of those skilled in the art to which this inventionpertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. The use of the word “a” or“an” when used in conjunction with the term “comprising” in the claimsand/or the specification may mean “one,” but it is also consistent withthe meaning of “one or more,” “at least one,” and “one or more thanone.” The use of the term “or” in the claims is used to mean “and/or”unless explicitly indicated to refer to alternatives only or thealternatives are mutually exclusive, although the disclosure supports adefinition that refers to only alternatives and “and/or.” Throughoutthis application, the term “about” is used to indicate that a valueincludes the inherent variation of error for the device, the methodbeing employed to determine the value, or the variation that existsamong the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. In one aspect such open ended terms alsocomprise within their scope a restricted or closed definition, forexample such as “consisting essentially of”, or “consisting of”.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof is intended to include atleast one of: A, B, C, AB, AC, BC, or ABC, and if order is important ina particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

All documents referred to herein are incorporated by reference to thefullest extent permissible.

Any element of a disclosure is explicitly contemplated in combinationwith any other element of a disclosure, unless otherwise apparent fromthe context of the application.

The present invention is further described by reference to the followingexamples, not limiting upon the present invention.

EXAMPLES Example 1 Cloning of Antibody Expression Vectors

The DNA expression constructs encoding the variable heavy (VH) andvariable light (VL) domains of an anti-TNFα antibody were previouslyprepared de novo and included restriction sites for cloning intomammalian expression vectors. Both heavy and light chain variable domainsequences were sequence optimised for expression in mammalian cells (formethodology see WO2009024567 and Kotsopoulou et al, J Biotechnol (2010)146: 186-193). Information describing the heavy and light chain variableregion sequences can be found in U.S. Pat. No. 6,090,382. To generatethe constructs used in this study, the variable heavy domain (VH)sequences were amplified using PCR. The PCR primers contained HindIIIand SpeI restriction sites to frame the VH domain containing the signalsequence for cloning into a pTT mammalian expression vectors containingthe human γ1 constant region. Similarly the VL domain sequence wasamplified by PCR using primers containing HindIII and BsiWI restrictionsites to facilitate cloning into a pTT mammalian expression vectorcontaining the human kappa constant region. The heavy chain expressionplasmid was given the code SJC322 and the light chain expression plasmidwas given the plasmid code SJC321.

DNA expression constructs encoding alternative variable heavy and lightchain regions of anti-TNFα antibodies with modifications in the CDRregions (as described in Rajpal et al. PNAS (2005) 102(24): pg8466-8471) were prepared de novo by build up of overlappingoligonucleotides and similar molecular biology techniques to thosedescribed above. The resulting plasmids encoding the heavy and lightchains of variants cb1-3, cb2-6 and cb2-44 are described in Table 1.

Example 2 Engineering of the Fc Region

Forward and reverse priming primers were used to introduce modifications(M252Y/S254T/T256E and T250Q/M428L) into the human γ1 constant region ofthe plasmid encoding the heavy chain of pascolizumab (anti-IL-4antibody) using the Quikchange protocol (Promega).

As described in Example 1 above, a PCR fragment encoding the VH domainof an anti-TNFα antibody was generated using a previously constructed,codon optimised vector as a template. The resulting fragment was clonedusing HindIII and SpeI into a pTT expression vector containing themodified human γ1 constant region described in the preceding paragraph.The plasmid encoding the heavy chain of the anti-TNFα antibody with theM252Y/S254T/T256E modification was designated SJC324. The plasmidencoding the heavy chain with the T250Q/M428L modification wasdesignated SJC323.

Forward and reverse priming primers were used to introduce modificationsinto the human γ1 constant region of anti-TNFα heavy chain expressionplasmid SJC322 using the Quikchange protocol (Promega). Plasmid SJC326encodes the anti-TNFα heavy chain containing the M428L/N434Smodification in the human γ1 constant region. Plasmid SJC328 encodes theanti-TNFα heavy chain containing the V308F modification in the human γ1constant region.

Example 3 Expression of Antibodies in HEK2936E Cells Using pTT5 EpisomalVectors

Expression plasmids encoding the heavy and light chains described abovewere transiently co-transfected into HEK 293 6E cells. Expressedantibody was purified from the supernatant by affinity chromatographyusing a 1 ml HiTrap Protein A column (GE Healthcare). Table 1 belowshows the list of antibodies produced.

Some antibodies were also expressed in CHO cells using a different setof expression vectors. See Examples 13, 14 and 15 for a description ofthe molecular biology, expression and purification.

TABLE 1 List of expressed antibodies Heavy SEQ Light SEQ chain ID ofchain ID of expression heavy expression light BPC code CDR variant Fcmodifications vector chain vector chain BPC1492 None Wild-type SJC322 12SJC321 2 BPC1494 None M252Y/S254T/T256E SJC324 5 SJC321 2 BPC1496 NoneM428L/N434S SJC326 9 SJC321 2 BPC1493 None T250Q/M428L SJC323 15 SJC3212 BPC1498 None V308F SJC328 18 SJC321 2 BPC1499 cb1-3 Wild-type SJC336150 SJC339 147 BPC1500 cb2-44 Wild-type SJC337 151 SJC340 148 BPC1501cb2-6 Wild-type SJC336 150 SJC338 149

Example 4 Binding of Antibodies to Tumour Necrosis Factor Alpha in aDirect Binding ELISA

A binding ELISA was carried out to test the binding of the expressedantibodies purified using protein A to recombinant tumour necrosisfactor alpha (TNFα). ELISA plates were coated with recombinant humanTNFα at 0.1 μg/ml and blocked with blocking solution (4% BSA. Variousdilutions of the purified antibody were added (diluted in 4% BSA in TTris-buffered saline at pH8.0 containing 0.05% Tween 20) and the platewas incubated for 1 hour at room temperature before washing in deionisedwater. Binding was detected by the addition of a peroxidase labelledanti human kappa light chain antibody (Sigma A7164) in blockingsolution. The plate was incubated for 1 hour at room temperature beforewashing in deionised water. The plate was developed by addition of OPDsubstrate (Sigma P9187) and colour development stopped by addition of 2MHCl. Absorbance was measured at 490 nm with a plate reader and the meanabsorbance plotted against concentration. The results are shown in FIG.1 and confirm that all the antibodies have a similar profile.

Example 5 Analysis of Antibodies in an L929 In Vitro NeutralisationAssay

This assay was used to test the neutralising ability of the antibodiesto neutralise TNF-α and inhibit cell death. Briefly, L929 cells wereseeded in a 96-well flat-bottomed plate at 10,000/well in 100 μl RPMI1640 (w/o phenol red) and incubated overnight at 37° C., 5% CO₂. Cellswere sensitised with 1.25 μg/ml actinomycin D for 1 hour. For theneutralising study, 0.001-60 μg/ml (0.0067-400 nM) anti-TNF-α mAb waspre-incubated with approx. 2 ng/ml (approximately 0.05 nM) TNF-α in a1:1 ratio for 1 hour at room temperature. For control group, RPMI wasused in place of the antibody. Following the 1 h pre-incubation withactinomycin D, 20 μl of antibody-antigen complex was added per well. 10μl media alone was added to wells as a negative control. Plates wereincubated at 18 hour at 37° C., 5% CO₂. Following this treatment period,cell viability was determined by a cell titer-Glo Luminescent assay kitaccording to manufacturer's instructions (Promega, Madison USA). ForL929 assay, the percentage cell viability of the unknowns was expressedas a percentage of the untreated group (taken as a 100%) and IC50 valueswere determined by Graphpad prism. Differences in IC50 values ofantibodies was assessed by one-way ANOVA (Newman-Keuls post hoc test)and considered significant at P-values of less than 0.05. Data isrepresented as mean±SEM, of n=4 experiments measured in duplicate. IC50values for each antibody were determined and are listed in Table 2below. The results show that the potency of all the antibodies testedare comparable.

TABLE 2 IC₅₀ values for various anti-TNFα antibodies in an L929neutralisation assay Antibody IC₅₀ value (μg/ml) BPC1492 1.19 ± 0.10BPC1494 1.20 ± 0.13 BPC1496 1.18 ± 0.10 Adalimumab 1.09 ± 0.07

Table 3 shows the IC50 values derived from the experiment. The resultsindicate that the improved anti-TNFα antibodies (BPC1499, BPC1500,BPC1501) show increased potency in this assay compared to BPC1492 andadalimumab.

TABLE 3 IC₅₀ values for improved anti-TNFα antibodies in an L929neutralisation assay Antibody IC₅₀ value (μg/ml) BPC1492 1.19 ± 0.1 BPC1499 0.21 ± 0.04 BPC1500 0.13 ± 0.02 BPC1501 0.21 ± 0.03 Adalimumab1.09 ± 0.07

Example 6 Effect of Antibodies on In Vitro IL-6 Release

The neutralising ability of antibodies was determined by measuring theireffect on inhibiting TNF-α mediated IL-6 release from whole blood cells.Briefly, 130 μL of whole blood was added to each well and plates wereincubated at 37° C. in a humidified 5% CO₂ incubator for 1 hour. For theneutralising study, 0.001-30 μg/ml (0.0067-200 nM) TNF-α mAb waspre-incubated with 10 ng/ml (approx. 0.4 nM) TNF-alpha in a 1:1 ratiofor 1 hour at 4° C. For control group, RPMI was used in place of theantibody. Following this pre-treatment, 20 μl of antigen-antibodycomplex or RPMI (negative control) was added per well and plates wereincubated for 24 hour at 37° C., 5% CO₂. 100 μL PBS (w/o MgCl₂ or CaCl₂)added to each well and placed on plate shaker for 10 mins at 500 rpm.Plates were then spun at 2000 rpm for 5 mins. 120 μL supernatant wascarefully removed and transferred to fresh 96-well round bottomed plateand IL-6 release was determined using an MSD based assay kit (Meso ScaleDiagnostics, Maryland USA). For the whole blood assay, the MSD signalfor each sample was read using a MSD SECTOR® Imager 2400 and IL-6release from the cells was quantified using a standard data analysispackage in PRISM 4.00 software (GraphPad. San Diego, USA). Thepercentage of IL-6 inhibition by each antibody was expressed as apercentage of the TNF-α alone treated group. Hence, dose response curveswere obtained for each antibody and IC50 values were determined. Usingthe log of the IC50 values, the difference in potency of the antibodieswas determined by one-way ANOVA (Newman-Keuls post hoc test) andconsidered significant at P-values of less than 0.05 for each donor(n=3). Data is represented as mean±SEM of three donors, measured induplicate. Table 4 below shows the IC50 values derived from these data.These results suggest that there is no significant difference in potencybetween the antibodies tested.

TABLE 4 IC₅₀ values for various anti-TNF antibodies in a TNFα-inducedIL-6 release assay Antibody IC₅₀ value (nM) BPC1492 0.72 ± 0.32 BPC14940.62 ± 0.11 BPC1496 0.64 ± 0.13 Adalimumab 0.47 ± 0.09

The IC50 values are shown in Table 5. The results indicate that theimproved anti-TNFα antibodies (BPC1499, BPC1500, BPC1501) show increasedpotency in this assay.

TABLE 5 IC₅₀ values for various improved anti-TNF antibodies in aTNFα-induced IL-6 release assay Antibody IC₅₀ value (nM) BPC1492 0.72 ±0.32 BPC1494 0.62 ± 0.12 BPC1499 0.14 ± 0.02 BPC1500 0.11 ± 0.05 BPC15010.15 ± 0.03 Adalimumab 0.47 ± 0.09

Example 7 Accelerated Stressor Studies

Prior to the study, antibodies to be tested were quantified on aspectrophotometer at OD280 nm and diluted to 1.1 mg ml in PBS (pH7.4).An aliquot was removed and 10% v/v of 500 mM sodium acetate was added togive a final concentration of 1 mg/ml at pH5.5 and the sample inspectedfor precipitation. The remaining sample in PBS had 10% PBS v/v added toa final concentration of 1 mg/ml at pH7.4 and an aliquot of this samplewas removed to provide a baseline aggregation level (as monitored bysize exclusion chromatography). The samples were then incubated at 37°C. for two weeks in an incubator, after which the samples werere-quantified on a spectrophotometer at OD280 nm and assessed (by sizeexclusion chromatography) for aggregation. The samples were tested forhuman TNFα binding in a direct binding ELISA. The results are shown inFIG. 2 and confirm that the binding activity of all antibodies tested iscomparable following the accelerated stressor study.

Example 8 Stability Study in 25% Human Serum

Prior to the study, antibodies to be tested were quantified on aspectrophotometer at OD280 nm and diluted to 1.25 mg/ml in PBS (pH7.4).An aliquot was removed and 25% v/v of human serum was added to give afinal concentration of 1 mg/ml. The remaining sample in PBS had 25% PBSv/v added to a final concentration of 1 mg/ml and an aliquot of thissample was removed to provide a baseline level. The samples were thenincubated at 37° C. for two weeks in an incubator, after which thesamples were tested for human TNFα binding in a direct binding ELISA.The results are shown in FIG. 3 and confirm that the binding activity ofall antibodies tested is comparable following incubation in 25% humanserum for two weeks.

Example 9 Analysis of Binding to Human TNFα Following Freeze-Thaw

Antibody samples were diluted to 1 mg/ml in a buffer containing 50 mMAcetate and 150 mM NaCl (pH6.0), snap-frozen in dry ice and then thawedat 4° C. overnight. Binding of the antibodies to human TNFα was testedin comparison to an antibody which had not been snap-frozen. To assessthe binding activity following freeze-thaw, ELISA plates were coatedwith recombinant human TNFα at 1 μg/ml and blocked with blockingsolution (4% BSA in Tris buffered saline). Various concentrations wereadded to the coated plates and incubated for 1 hour at room temperaturebefore washing in deionised water. Binding was detected by the additionof a peroxidase labelled anti human kappa light chain antibody (SigmaA7164) in blocking solution. The plate was incubated for 1 hour at roomtemperature before washing in deionised water. The plate was developedby addition of OPD substrate (Sigma P9187) and colour developmentstopped by addition of 2M HCL. Absorbance was measured at 490 nm with aplate reader and the mean absorbance plotted against concentration. Theresults are shown in FIG. 4 and confirm that the binding activity of allantibodies tested is comparable following freeze-thaw.

Example 10 Analysis of Binding of Anti-TNFα Antibodies to FcγRIIIa

ELISA plates were coated with recombinant human FcγRIIIa (V158 and F158variants) at 1 μg/ml and blocked with blocking solution (4% BSA in Trisbuffered saline). Various concentrations were added to the coated platesand incubated for 1 hour at room temperature before washing in deionisedwater. Binding was detected by the addition of a peroxidase labelledanti human kappa light chain antibody (Sigma A7164) in blockingsolution. The plate was incubated for 1 hour at room temperature beforewashing in deionised water. The plate was developed by addition of OPDsubstrate (Sigma P9187) and colour development stopped by addition of 2MHCl. Absorbance was measured at 490 nm with a plate reader and the meanabsorbance plotted against concentration. The results are shown in FIGS.5 a and 5 b and confirms that BPC1494 has reduced capacity to bindFcγRIIIa (V158 and F158 variants) compared to BPC1492 and BPC1496.

Example 11 PreteOn Analysis: FcRn Binding

Antibodies for testing were immobilised to similar levels on a GLCbiosensor chip (BioRad 176-5011) by primary amine coupling. Recombinanthuman and cynomolgus FcRn were used as analytes at 2048 nM, 512 nM, 128nM, 32 nM, and 8 nM, an injection of buffer alone (i.e. 0 nM) was usedto double reference the binding curves. Regeneration of the antibodysurface following FcRn injection used HBS-N at pH9.0, the assay was runon the PrateOn XPR36 Protein Interaction Array System at 25° C. and runin HBS-N pH7.4 and HBS-N pH6.0 with the FcRn diluted in appropriatebuffer. Affinities were calculated using Equilibrium model, inherent tothe PrateOn analysis software, using a “Global R-max” for binding atpH6.0 and the R-max from binding at pH6.0 for affinity calculation atpH7.4. Since the binding curves did not reach saturation at pH7.4, thevalues obtained are unlikely to be true affinities however they can beused to rank constructs. The results are shown in Table 6 and confirmthat BPC1494 and BPC1496 have an improved affinity for human and cynoFcRn at pH6.0 when compared to BPC1492.

TABLE 6 Affinities of Anti-TNF alpha constructs binding to Human andCyno FcRn BPC Human pH 6.0 Human pH 7.4 Cyno pH 6.0 Cyno pH 7.4 NumberKD(nM) KD(nM) KD(nM) KD(nM) BPC1492 554 21200 579 29700 BPC1494 204 2320239 2640 BPC1496 144 1910 154 2100 BPC1497 428 15500 464 20800 BPC1498357 5910 402 6280 BPC1493 264 4390 295 4690

Example 12 PK Studies in Human FcRn Transgenic Mice

In a single dose pharmacokinetic study BPC1494 and BPC1492, wereadministered intravenously (IV) at 1 mg/kg to two different strains ofFcRn humanised mice and one strain deficient in FcRn (Petkova et al.Int. Immunol (2010) 18(12): 1759-1769). Plasma samples were analyzed forBPC1494 or BPC1492, as appropriate, using a validated Gyrolabfluorescent immunoassay.

The methods used biotinylated human TNF alpha as the capture antigen andan Alexa labelled anti-human IgG (Fc specific) antibody as the detectionantibody. Using an aliquot of mouse plasma diluted 1:10 with assaybuffer, the lower limit of quantification (LLQ) was 100 ng/mL and thehigher limit of quantification (HLQ) was 100,000 ng/mL. Plasmaconcentrations below the lowest standards were considered to be notquantifiable. QC samples prepared at three different concentrations andstored with the study samples, were analysed with each batch of samplesagainst separately prepared calibration standards. For the analyses tobe acceptable, at least one QC at each concentration must not deviatefrom nominal concentration by more than 20%. The QC results from thisstudy met these acceptance criteria.

PK analysis was performed by non-compartmental pharmacokinetic analysisusing WinNonLin, version 6.1. All computations utilised the nominalblood sampling times. The systemic exposure to BPC1494 and BPC1492 wasdetermined by calculating the area under the plasma concentration timecurve (AUC) from the start of dosing until the last quantifiable timepoint (AUC_(0-t)) using the linear log trapezoidal calculation method.Further PK parameters could not be derived from the data duediscrepancies in sample labelling.

TABLE 7 Summary pharmacokinetic parameters for BPC1494 and BPC1492following a single intravenous administration (bolus) at a target doseof 1 mg/kg to transgenic mice Compound Strain Cmax (ug/mL) AUC(hr*ug/mL) BPC1494 1 13.8 2240 BPC1492 14.8 1730 BPC1494 2 12.0 1320BPC1492 13.2 1060 BPC1494 3 13.6 214 BPC1492 12.2 250 Strain 1 =mFcRn−/− hFcRn (32) Tg/Tg Strain 2 = mFcRn−/− hFcRn (276) Tg/Tg Rag1−/−Strain 3 = mFcRn −/−/Rag1−/− Similar C_(max) concentrations wereobtained for all groups. In both human FcRn knock-in mouse strainsBPC1494 had a higher exposure (AUC_(0-t)) than BPC1492, although thisdifference was not notable (1.3 fold). In the absence of both human andmouse FcRn BPC1492 had a higher exposure than BPC1494.

Example 13 Cloning of Antibody Expression Vectors into pEF Vectors

In some cases, the DNA encoding the expression cassettes for the heavyand light chains were excised from the vectors described in Example 3using HindIII and EcoRI and cloned into pEF vectors, where expressionoccurs from the hEF1a promoter, using standard molecular biologytechniques (for description of vectors see Kotsopoulou et al J.Biotechnol (2010) 146: 186-193).

TABLE 8 Heavy Light Heavy Light chain chain chain chain BPC Fcexpression expression SEQ SED code modification vectors vector ID No. IDNo. BPC1492 None SJC330 SJC329 12 2 BPC1494 M252Y/S254T/ SJC331 SJC329 52 T256E BPC1496 M428L/N434S SJC332 SJC329 9 2

Example 14 Expression of Antibodies in CHO Cells Using pEF ExpressionVectors

Expression plasmids encoding heavy and light chains were co-transfectedinto CHO DG44 cells and expressed at scale to produce antibody. For thegeneration of BPC1492 plasmids SJC329 and SJC330 were used. For theexpression of BPC1494 plasmids SJC329 and SJC331 were used. For BPC1496plasmids SJC329 and SJC332 were used.

Briefly, 30 μg DNA (15 μg heavy chain and 15 μg light chain) waslinearised overnight with Not1 restriction enzyme. The resultantrestricted DNA was then ethanol precipitated and re-dissolved in TEbuffer. From culture, 6×10⁶ CHO DG44 cells were obtained and washed in10 ml of PBS. The cell pellet was then re-suspended in 300 μl of Amaxasolution V. 100 μl of the aforementioned cell suspension was then addedinto to each of three Amaxa cuvettes, which also contained 3 μg of thelinearised DNA. The cuvettes were inserted into an Amaxa nucleofector IIdevice and electroporated with pre-set programme U-023. The contents ofthe three cuvettes (300 μl) of electroporated cells were added to 10 mlof warmed MR14 medium (including nucleosides and BSA) and incubated in aT75 flask for 48 hours. Following this period, the medium was changed tonucleoside-free-MR14 (MR14 containing only BSA)). Every 3-4 days,conditioned medium was removed and replaced with fresh selection medium.Once cells had undergone recovery, the medium was substituted to 2×MR14and IgG expression was confirmed by nephlometry. 2 L shake-flasks wereseeded with 1 L of the IgG-expressing cells at 0.6×10⁶/ml and grown for7 days. Cells were separated from supernatant by centrifugation and thesupernatant was used for protein purification.

1 litre cell culture supernatants were purified using a 2-step automatedprocess on an AKTA Xpress system. The antibody was captured on a 5 mlMabSelectSure column and then washed prior to elution. The elutedantibody was then loaded onto a 440 ml Superdex 200 gel filtrationcolumn and 2 ml fractions collected in a 96-well block. Fractions ofpurified antibody were pooled and 0.2 μm filtered and then concentratedto ˜5 mg/ml using Amicon spin concentrators. The final material wasagain 0.2 μm filtered and then dispensed into sterile tubes fordelivery. The final material was subject to analytical SEC to determineaggregation, an endotoxin assay, LC-MS for accurate mass determination(included PNGaseF and untreated material to determine glycosylation),SDS PAGE electrophoresis, PMF for sequence confirmation and A280 forconcentration determination.

Example 15 Alternative Method for Expression of Antibodies in CHO CellsUsing pEF Expression Vectors

DHFR-null CHO DG44 cells were obtained from Dr. Chasin of ColumbiaUniversity. These cells were subsequently adapted to a chemicallydefined medium. These adapted host cells were designated DG44-c and arecultured in proprietary chemically defined medium supplemented withGlutamax and HT-supplement.

Generation of the polyclonal pool: For more details on protocols seeWO2009024567 and Kotsopoulou et al, J. Biotechnol (2010) 164(4):186-193. Briefly, DG44-c cells were transfected with plasmids encodingthe heavy and light chains and DHFR and neoR respectively byelectroporation (using the Amaxa nucleofector system). At 48 hours posttransfection, selection was initiated by addition of G418 (at a finalconcentration of 400 μg/ml) and removal of HT. When viability and cellcounts increased sufficiently (in this case 2 months post transfection)methotrexate (MTX) was added at a final concentration of 5 nM. Cellswere scaled up and production curves were initiated 9-16 days afteraddition of MTX. For these production curves cells were seeded at0.6-0.8×10⁶ cells/ml in chemically defined media and were fed on days 6,9 or 10, 12 or 13 and/or 16. Supernatant was collected when viabilitydropped to approximately 50% and the cells were removed bycentrifugation at 4000 g for 30 mins followed by filtration through asartobran capsule.

Antibodies were purified at room temperature using a two stepchromatographic procedure: Initial capture was performed using a 50 mlMabSelect SuRe column (GE Healthcare) followed by Size ExclusionChromatography (SEC) with a 1.5 L Superdex 200 μg SEC (GE Healthcare).The conditioned media was loaded onto a pre-equilibrated MabSelect SuRecolumn at a flow rate of 9 cm/h. Following washing to base line withequilibration buffer (50 mm Tris pH 8.0, 2M NaCl) the column was washedwith a low salt buffer buffer (50 mM NaCl Tris pH 8.0, 150 mM NaCl)until conductivity was stable. The column was then eluted with elutionbuffer (25 mM Citrate pH 2.5). Fractions corresponding to peak proteinelution were immediately neutralized with 1/10 vol. 1.0M Tris pH 8.0which were then pooled and filtered through a 0.2 μm bottletop filter.The recovered sample was loaded at 21 cm/h onto the SEC columnpre-equilibrated with SEC buffer (50 mM Na Acetate, 150 mM NaCl). Thefractions containing the main (monomeric) protein peak were pooled andfilter sterilized.

Antibodies prepared by this method were used for analyticalcomparability studies summarised in the following example.

Examples 16 Analytical Comparability on Stressed and Control Samples

Size exclusion chromatography was carried out to determine theaggregation levels of the protein. The optimised method involvedinjection of the sample onto a TOSOH TSK G3000SWXL column which had beenequilibrated in 100 mM sodium phosphate, 400 mM NaCl, pH 6.8. Absorbancewas measured at both 280 nm and 214 nm. Reverse-phase HPLC separatesproteins and their isoforms based on hydrophobicity. Protein wasinjected onto a PLRP-S 1000° A 8 μm column and eluted using a gradientproduced by 50% Formic acid, and 95% Acetonitrile. Absorbance wasmeasured at 280 nm. The purity of the molecule is reported as apercentage of the main peak area relative to the total peak area.Different isoforms of the mAb were separated on the basis of their pIvalues using capillary isoelectric focussing (cIEF). IEF separation wasperformed on a 10 cm, UV280 transparent cartridge capillary. Theoptimised method involved a solution containing 5% pH 3-10 ampholytes,10 mM NaOH, protein of interest and internal pI markers (7.05 and 9.5)which was loaded into the capillary by pressure injection.

The specific activity of antibodies (adalimumab, BPC1494, BPC1496) wasdetermined using MSD. In brief, 96-well plates were coated with 50 μLper well TNFα diluted to 1 μg/mL in PBS. The plate was incubated on thebench top at ambient temperature without shaking for 2 hours. Thecoating solution was removed and the plate was blocked with 50 μL perwell of 1% BSA in PBS, with 0.05% Polysorbate 20. The plate wasincubated for 1 hour at 24° C. with shaking at 400 rpm and then washed 4times with wash buffer. The antibodies were diluted in 0.1% BSA in PBSwith 0.05% Polysorbate 20 and 30 μl of each sample was added to theplate. The plate was incubated for 1 hour at 24° C. with shaking at 400rpm. The plate was then washed 4 times with wash buffer. Anti-human IgGsulfotag was diluted 1 in 5000 in assay buffer. 30 μL was added to eachwell of the plate and then incubated for 1.5 hour at 24° C., withshaking at 400 rpm. The plate was then washed 4 times with wash buffer.The 4×MSD Read Buffer concentrate was diluted to 1× using deionisedwater. 100 μL was then added per well of the plate. The plate was thenread using the MSD Sector Imager instrument. From the signals obtainedfrom the assay, specific activities of the molecules were calculated.

Deamidation Analysis

Deamidation is a common post-translational modification that can occurto asparagine and glutamine residues, but is most commonly observed withasparagine residues, particularly when adjacent to a glycine residue. Inorder to examine how susceptible these residues are and to determine theeffects of deamidation on potency, adalimumab, BPC1494 and BPC1496 wereexposed to a stress study. The stress was carried out by incubation in1% ammonium bicarbonate at pH 9.0, for 48 hrs, conditions which havepreviously been shown to cause deamidation. The stressed samples wereincubated alongside a control (in PBS) and were compared to this as wellas an unstressed reference and analysed using c-IEF, SEC and BindingELISA. Forced deamidation was also done on all samples in the presenceand absence of EDTA. It has been shown previously that forceddeamidation conditions cause fragmentation in addition to deamidation.EDTA prevents and or minimizes the fragmentation.

Oxidation Analysis

Oxidation of various residues can occur throughout the processing andstorage of proteins; however the most commonly oxidised residue ismethionine, which was the focus of this screen. Oxidation susceptibilityof these residues was examined through exposure to stress conditions byincubation in 5 mM and 50 mM H₂O₂ for 30 minutes and evaluated usingRP-HPLC, SEC and ELISA.

Summary of Results

Both BPC1494 and BPC1496 behave very favourably compared to adalimumabas shown by analytical comparability on both stressed and controlsamples. For all antibodies tested, no significant degradation wasobserved under forced oxidation conditions as shown by all analyticaltechniques employed. Significant deamidation as measured by c-IEF wasobserved at pH 9.0 as expected for all antibodies tested. In addition wesaw significant fragmentation for all antibodies tested as shown by SECat pH 9.0 in samples without EDTA, this is also as expected. There is areduction in the pI value, (approximately 0.2) of BPC1494 when comparedto adalimumab. This is attributed to the presence of an additionalglutamic acid residue in the heavy chain sequence of the BPC1494 thusmaking it more acidic. Forced deamidation and oxidation had minimalimpact on binding and this was observed for BPC1494, BPC1496 andadalimumab.

Example 17 Analysis of Binding of Improved Antibodies by ELISA

Antibodies BPC1499, 1500 and 1501 were assessed for binding activity byELISA as described in Example 4. Using two different antigen coatingconcentrations (0.1 and 1.0 μg/ml), the antibodies did not show anydifference in their binding profile when compared with BPC1492. Underthe conditions tested, it appears that the ELISA does not discriminatebetween antibodies with different reported binding activities. The sameantibodies were assessed using methodologies described in Examples 18, 5and 6 which are considered more sensitive assays. In these assays,antibodies BPC1499, 1500 and 1501 show improved binding affinity andimproved potency when compared with BPC1492.

Example 18 Biacore Analysis of TNF Alpha Binding Using a Capture Surface

Protein A and anti-human IgG (GE Healthcare BR-1008-39) were coupled onseparate flow cells on a CM3 biosensor chip. These surfaces were used tocapture the antibodies for binding analysis. Recombinant human andcynomolgus TNF alpha were used as analytes at 64 nM, 21.33 nM, 7.11 nM,2.37 nM, 0.79 nM, an injection of buffer alone (i.e. 0 nM) used todouble reference the binding curves. Regeneration of the capture surfacewas carried out using 100 mM phosphoric acid and 3M MgCl₂. The run wascarried out on the Biacore T100 machine at 37° C. using HBS-EP asrunning buffer. The constructs BPC1494 and BPC1496 showed reducedbinding to Protein A and the anti-human IgG surface making thesesurfaces unsuitable for generating kinetics for those molecules.

TABLE 9 Kinetic Analysis of Human and Cyno TNF alpha Binding to CapturedAnti-TNF alpha Antibodies. Construct Analyte Capture Surface ka(1/Ms)kd(1/s) KD(nM) BPC1492 human TNFα Protein A 2.12E+06 1.10E−04 0.05196BPC1494 human TNFα Protein A Data not Analysable BPC1496 human TNFαProtein A Data not Analysable BPC1500 human TNFα Protein A 2.68E+064.19E−05 0.01561 BPC1492 human TNFα anti-human IgG 6.78E+06 1.73E−040.02554 BPC1494 human TNFα anti-human IgG Data not Analysable BPC1496human TNFα anti-human IgG Data not Analysable BPC1500 human TNFαanti-human IgG 4.51E+06 7.07E−05 0.01568 BPC1492 Cyno TNFα Protein A1.10E+06 1.11E−04 0.101  BPC1494 Cyno TNFα Protein A Data not AnalysableBPC1496 Cyno TNFα Protein A Data not Analysable BPC1500 Cyno TNFαProtein A 2.34E+06 3.51E−05 0.01503 BPC1492 Cyno TNFα anti-human IgG1.96E+06 3.75E−04 0.1911  BPC1494 Cyno TNFα anti-human IgG Data notAnalysable BPC1496 Cyno TNFα anti-human IgG Data not analysable BPC1500Cyno TNFα anti-human IgG 4.48E+06 2.09E−04 0.04667

Example 19 ProteOn Reverse Assay Binding Analysis

Biotinylated TNF alpha was mixed with biotinylated BSA at a 1:49 ratio,at a final total protein concentration of 20 μg/ml (i.e. 0.4 μgbiotinylated TNF alpha and 19.6 μg biotinylated BSA). This mixture wascaptured on a NLC biosensor chip (a single flowcell) (Biorad 176-5021).The chip surface was conditioned with 10 mM glycine pH3.0 till a stablesignal was achieved. The antibodies to be tested were used as analytesat 256 nM, 64 nM, 16 nM, 4 nM and 1 nM and 0 nM. The binding curves werereferenced against a flowcell coated with biotinylated BSA alone.Regeneration was achieved using 10 mM glycine pH3.0. Data was fitted tothe 1:1 model inherent to the PrateOn analysis software.

TABLE 10 Apparent Kinetics of Anti-TNF alpha antibodies binding toNeutravidin Captured TNF alpha BPC Number ka (1/Ms) kd (1/s) KD (nM)BPC1499 2.27E+06 1.72E−05 0.008 BPC1500 2.06E+06 3.00E−05 0.015 BPC15011.17E+06 6.97E−05 0.06 BPC1496 6.33E+05 4.04E−04 0.639 BPC1494 7.23E+053.50E−04 0.484 BPC1492 7.89E+05 3.21E−04 0.407

This data is one set of two experiments which were carried out (secondset not shown). The KD ranking of the data is representative of bothdata sets.

Example 20 Construction of Alternative Antibodies which Bind to HumanTNFα

The DNA expression constructs encoding additional variable heavy regionswith modifications in the CDR regions (as described in Rajpal et al.PNAS (2005) 102(24): pg 8466-8471) were prepared de novo by build up ofoverlapping oligonucleotides and similar molecular biology techniques tothose described in Example 1. Examples of DNA sequences encoding thevariable heavy domains of these variant antibodies are given in SED IQNO: 81, 83, 85, 87, 89, 91, 93 and 95. The DNA expression constructsencoding additional variable light domain regions with modifications inthe CDR regions (as described in Rajpal et al. PNAS (2005) 102(24): pg8466-8471) were prepared de novo by build up of overlappingoligonucleotides and similar molecular biology techniques to thosedescribed in Example 1. Examples of DNA sequences encoding the variablelight domains of these variant antibodies are given in SED IQ NO: 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135 and 137. Once constructed, the expressionplasmids encoding the heavy and light chains were transientlyco-transfected into HEK 293 6E cells. Expressed antibody were purifiedfrom the supernatant and assessed for activity using the methods similarto those described in Example 6.

Example 21 Construction of Expression Vectors for BPC2604(Pascolizumab-YTE)

The pTT-based DNA expression constructs encoding the heavy chain ofpascolizumab was engineered to include the following changesM252Y/S254T/T256E (EU index numbering) using the Quikchange protocol(Promega).

Example 22 Expression/Purification of Pasco and Pasco-YTE Vectors

Expression plasmids encoding the heavy and light chains of BPC2604 weretransiently co-transfected into HEK 293 6E cells. Expressed antibody waspurified from the bulk supernatant using a two step purification carriedout by affinity chromatography and SEC using a 5 ml MabSelectSure columnand Superdex 200 column on an AKTA Xpress.

Example 23 BIAcore Analysis of Pasco Vs. Pasco YTE for FcRn Binding

Antibodies were immobilised on a GLM chip (20 μg/ml in acetate pH4.5) byprimary amine coupling. Human, cynomolgus, rat and mouse FcRn receptorsused at 2048, 512, 128, 32 and 8 nM. 0 nM used for double referencing.Assay were carried out in HBS-EP pH7.4 and HBS-EP pH6.0 (FcRn receptordiluted in appropriate running buffer for each pH. The surface wasregenerated for FcRn binding with 200 mM Tris pH9.0. Data was fitted toan equilibrium model, with R-max set to highest R-max obtained of anyconstruct. The results are shown in Table 11 below and confirm that theYTE-modified pascolizumab (BPC2604) shows improved binding to FcRn atpH6.0 compared to pascolizumab.

TABLE 11 Affinities of anti-IL-4 antibody constructs for Human and CynoFcRn (n.a.b. is no analysable binding) KD (nM) at pH 6.0: KD (nM) at pH7.4: R-max = 1020 R-max = 1020 Human Cyno Mouse Rat Human Cyno Mouse RatAntibody Fc modification FcRn FcRn FcRn FcRn FcRn FcRn FcRn FcRn BPC2604M252Y/S254T/T256E 98 92.1 53.4 66.0 11600 11100 2160 4330 PascolizumabNone 541 505 205 228 n.a.b n.a.b n.a.b n.a.b

Example 24 PK Studies with Pasco Vs. Pasco-YTE

FIG. 6 shows the average dose normalised plasma concentrations ofpascolizumab-YTE (BPC2604)) in female cynomolgus monkeys andpascolizumab in male cynomolgus monkeys following a single intravenous(1 hr infusion) administration at a target dose of 1 mg/kg. The data forBPC2604 and pascolizumab were generated in separate studies. Plasmaantibody concentrations for pascolizumab and BPC2604 were assessed bychemi-luminescence ELISA using IL-4 as the capture reagent andanti-human IgG (Fc specific)-HRP conjugate as the detection reagent. Thevalidated range for the assay was 50-5000 ng/mL. The results are shownin FIG. 6. Both compounds had similar Cmax but BPC2604 had a 3-foldlower plasma clearance resulting in 3-fold increase in AUC and 2-foldincrease in half-life (T½).

Example 25 Formulation Studies at 5 mg/ml

The stability of adalimumab and the TNF-alpha variant BPC1494 in twoformulations was compared. Formulation ‘A’ (citrate-phosphate buffer) isthe marketed adalimumab formulation made up of 6.16 mg/ml Sodiumchloride+0.30 mg/ml Sodium citrate monobasic+1.30 mg/mL Citric acidmonohydrate+12 mg/ml Mannitol+0.86 mg/mL Monobasic sodium phosphatedihydrate+1.53 mg/mL Dibasic sodium phosphate dihydrate+1.0 mg/ml PS80at pH 5.2.

Formulation ‘B’ (acetate buffer) is composed of 6.81 mg/mL (50 mM)Sodium Acetate trihydrate+10 mg/mL (1% w/v) Arginine+0.0186 mg/mL (0.05mM) EDTA+2.98 mg/mL (51 mM) Sodium Chloride+0.2 mg/mL (0.02% w/v)Polysorbate 80, adjusted to pH 5.5 using HCl or NaOH.

The TNF-alpha variant BPC1494 material used in this study was made in aChinese Hamster Ovary (CHO DG44) cell line and purified using a two stepprocess involving mAb Select Sure followed by Superdex column 200 μg.Adalimumab (Product code NDC 0074-3799-02, Lot number 91073LX40)manufactured by Abbott Laboratories was used.

Adalimumab was re-formulated into Formulation ‘B’ by overnight dialysisat 5° C. using a 10 KDa Slide—A—Lyzer cassette (Product Number 66830,Lot Number LJ150514); produced by Thermo Scientific (Rockford, Ill.;USA). This experiment was carried out at a different time point to theother three formulations. Both Adalimumab in Formulations ‘A’ and ‘B’were diluted to 5 mg/mL using their respective formulation buffers. TheTNF-alpha variant BPC1494 molecule was also formulated in Formulations Aand B at ˜5 mg/mL. A total of 4 samples were filtered through a MillexGV0.22 um filter under a clean laminar flow condition before beingtransferred into labelled pre-sterilized glass vials and incubated at 5°C., 25° C. and 40° C. for up to 14 weeks. Samples were taken at selectedtime points and analysed using SEC-HPLC (Table 12), cIEF (Table 13).Other assays as described below were also carried out to assess thestability of the antibodies.

Appearance by Visual Observation

Samples were inspected for clarity under daylight conditions. Bothantibodies in each formulation remained unchanged (clear colourlesssolution) after 14 weeks storage at 5° C., 25° C. and 40° C.

Protein Concentration (A280 nm) Measurement

Protein concentration was measured using a nanodrop spectrometer, whichis indicative of protein stability. The extinction coefficient foradalimumab is 1.46 and for TNF-alpha variant BPC1494 is 1.48. There wasno significant difference in the results after 14 weeks storage at 5°C., 25° C. and 40° C.

pH

pH was measured for all samples stored under different storageconditions to determine whether any significant pH drifts had occurred.All results remained within assay variability after 14 weeks storage at5° C., 25° C. and 40° C.

Size Exclusion Chromatography (SEC)

This method separates soluble protein molecules in the solution based onsize and not molecular weight. In theory, small molecules will penetrateevery small pore of the stationary phase and hence will elute later. Thechromatogram obtained enables the determination of percentage area ofaggregates, monomer and low molecular weight (MW) fragments. Thepresence of aggregates and/or low molecular weight species is indicativeof protein degradation. Increased stability corresponds to a highpercentage of monomeric species (Mono) together with a low percentage ofTotal Aggregates (TA) and Total Low Molecular weight Fragments (TLMWF).

SEC-HPLC data (Table 12) shows that the TNF-alpha variant BPC1494 wasrelatively more stable in formulation ‘B’ compared to formulation Aafter storage at 25° C. and 40° C. for 14 weeks. Furthermore, TNF-alphavariant BPC1494 was relatively more stable, or at least as stable asadalimumab in formulation A. The results for adalimumab in formulation Bare all within 5% TA and/or TLMWF. Therefore, formulation B hasadvantages over formulation A for both TNF-alpha variant BPC1494 andadalimumab.

For example, Table 12 shows that after storage at 25° C. for 8 weeks,TNF-alpha variant BPC1494 in formulation A has 2.3% TLMWF whileformulation B produced only 1.5%. Furthermore, TNF-alpha variant BPC1494in formulation B was relatively more stable than adalimumab informulation A (1.8% TLMWF). Similarly, at the 14 week time point at 25°C., 3.15% TLMWF was observed for TNF-alpha variant BPC1494 informulation ‘A’ compared to 2.3% TLMWF in formulation ‘B’. Furthermore,TNF-alpha variant BPC1494 in ‘B’ was relatively more stable thanadalimumab in formulation A (3.4% TLMWF). A similar trend for TLMWF wasobserved for both molecules on incubation at 40° C. for 4 weeks(adalimumab in ‘A’: 3.6%; TNF-alpha variant BPC1494 in ‘A’: 4.1%;TNF-alpha variant BPC1494 in ‘B’: 2.6%).

Also, results for Total Aggregate (TA) show that at 14 weeks at 25° C.,the TNF-alpha variant BPC1494 was relatively more stable in ‘B’ (0.3%)than in ‘A’ (0.5%); and relatively more stable than adalimumab informulation A (0.4%).

Capillary Iso-Electric Focusing (c-IEF)

This technique is used for determining the charge profile of molecules.A broad pI range reflects greater charge heterogeneity of the Productand in addition a broad pI range may be indicative of degradation.Typically the number of peaks will increase with increased degradation.The C-IEF data of Table 12 supports the SEC findings in Table 13.

The % area of main isoform (% AMI) was comparable between adalimumab informulation A and TNF-alpha variant BPC1494 in formulation B at Weeks 8and 14 at 25° C. (56.0-57.7 and 53.2 respectively). At these time pointsand temperature, formulation B shows a slight advantage over ‘A’ forTNF-alpha variant BPC1494.

Similarly, adalimumab is relatively more stable in formulation ‘B’ thanin formulation ‘A’ (see Week 4 data). For example, increased changes incharge heterogeneity (i.e. increase in number of peaks) were observedfor adalimumab incubated for up to 4 weeks at 40° C. in formulation ‘A’compared to formulation ‘B’ (8 peaks and 6 peaks respectively).TNF-alpha variant BPC1494 showed a more consistent charge heterogeneityof 5 peaks at all timepoints and temperatures.

Functional Binding Assay

The binding activity of adalimumab and TNF-alpha variant BPC1494 in thetwo formulations was assessed by Biacore. Over a 14 week period ofstorage at 5° C., 25° C. and 40° C., the samples showed similar %binding within assay variability.

Hence, it can be concluded that formulation ‘B’ can serve as analternative to formulation ‘A’ in a clinical setting withoutcompromising the stability of the protein and potentially eliminatingthe pain associated with the marketed adalimumab formulation (A).

Importantly, this data shows that not only does the acetate formulation(B) improve the stability of the TNF-alpha variant BPC1494 compared tothe citrate-phosphate formulation (A); but the acetate formulation iscomparable or slightly better than the citrate-phosphate formulationwhen stabilising adalimumab.

TABLE 12 SEC-HPLC of adalimumab and TNF-alpha variant BPC1494 inFormulation ‘A’ and ‘B’ at 5° C., 25° C. and 40° C. TLMWF: Total LowMolecular Weight Fragment; Mono: Monomer; TA: Total Aggregate. N = 2Condition Initial Week 2 Week 4 Week 8 Week 14 ° C. TA Mono TLMWF TAMono TLMWF TA Mono TLMWF TA Mono TLMWF TA Mono TLMWF adalimumab informulation ‘A’  5° C. 0.30 99.57 0.13 NT 0.27 99.58 0.15 0.32 99.480.19 0.49 99.28 0.23 25° C. 0.23 99.55 0.22 0.20 99.57 0.23 0.30 97.871.83 0.43 96.16 3.41 40° C. 0.24 96.86 2.91 0.29 96.06 3.65 NT NTadalimumab in formulation ‘B’  5° C. 0.34 99.47 0.18 NT 0.23 99.46 0.310.23 99.53 0.24 NT 25° C. NT NT NT NT 40° C. 0.22 97.60 2.18 0.24 95.74.06 0.34 94.92 4.74 NT TNF-alpha variant BPC1494 in formulation ‘A’  5°C. 0.28 99.72 0.00 NT 0.27 99.73 0.00 0.31 99.59 0.10 0.44 99.41 0.1525° C. 0.28 99.59 0.13 0.28 98.50 1.22 0.39 97.32 2.30 0.47 96.38 3.1540° C. 0.34 96.71 2.96 0.85 95.09 4.07 NT NT TNF-alpha variant BPC1494in formulation ‘B’  5° C. 0.29 99.71 0.00 NT 0.27 99.73 0.00 0.28 99.530.19 0.29 99.59 0.12 25° C. 0.27 99.58 0.15 0.26 99.62 0.12 0.30 98.161.54 0.30 97.39 2.31 40° C. 0.28 99.40 0.31 0.25 97.17 2.58 NT NT NT =Not Tested

TABLE 13 CE-IEF of adalimumab and TNF-alpha variant BPC1494 inFormulation ‘A’ and ‘B’ at 5° C., 25° C. and 40° C. N = 2 Initial Week 2Week 4 Condition % % % ° C. pI R pMI AMI No P pI R pMI AMI No P pI R pMIAMI No P adalimumab in formulation ‘A’  5° C. 8.52-8.98 8.72 62.8 6 NT8.52-8.96 8.72 62.2 6 25° C. 8.58-9.05 8.81 52.4 6 8.50-8.96 8.72 60.3 640° C. 8.53-9.06 8.79 41.4 8 8.49-9.05 8.71 43.3 8 adalimumab informulation ‘B’  5° C. 8.57-9.07 8.79 60.5 6 NT 8.55-9.02 8.76 59.9 6 25C NT NT 40° C. 8.53-9.02 8.75 53.2 6 8.53-9.02 8.75 47.9 6 TNF-alphavariant BPC1494 in formulation ‘A’  5° C. 8.18-8.64 8.50 57.6 5 NT8.19-8.64 8.50 60.2 5 25° C. 8.20-8.69 8.53 57.7 5 8.19-8.62 8.50 57.9 540° C. 8.21-8.70 8.54 50.0 5 8.00-8.60 8.50 37.0 5 TNF-alpha variantBPC1494 in formulation ‘B’  5° C. 8.19-8.65 8.50 58.3 5 NT 8.20-8.658.51 59.3 5 25° C. 8.22-8.70 8.54 57.8 5 8.20-8.65 8.51 57.1 5 40° C.8.22-8.70 8.54 50.7 5 8.01-8.62 8.51 38.0 5 Week 8 Week 14 Condition % %° C. pI R pMI AMI No P pI R pMI AMI No P adalimumab in formulation ‘A’ 5° C. 8.53-9.00 8.74 62.2 6 8.48-8.95 8.71 60.0 5 25° C. 8.51-9.00 8.7457.7 6 8.51-8.98 8.73 53.2 5 40° C. NT NT adalimumab in formulation ‘B’ 5° C. 8.49-8.96 8.72 61.0 5 NT 25 C NT NT 40° C. 8.49-9.07 8.72 39.8 6NT TNF-alpha variant BPC1494 in formulation ‘A’  5° C. 8.21-8.67 8.5159.1 5 8.17-8.62 8.49 58.8 5 25° C. 8.20-86.6 8.51 54.5 5 8.17-8.61 8.4952.1 5 40° C. NT NT TNF-alpha variant BPC1494 in formulation ‘B’  5° C.8.21-8.67 8.52 59.4 5 8.18-8.65 8.50 58.5 5 25° C. 8.21-8.67 8.52 56.0 58.18-8.64 8.50 53.2 5 40° C. NT NT NT = Not Tested; pI R: Pi Range; pMI:pI of Main Isoform; % AMI: % Area Main Isoform; NoP: Number of Peaks.

Example 26 Formulation Studies at 50 mg/ml

As shown in the previous example 25, adalimumab and TNF-alpha variantBPC1494 at 5 mg/mL in formulation ‘B’ can serve as an alternative toformulation ‘A’. This example is focused on comparing the stability ofadalimumab in its marketed formulation ‘A’ compared to formulation ‘B’and other TNF-alpha variants at 50 mg/ml.

Two samples of TNF-alpha variant BPC1494 were analysed, one expressed inCHO DG44 cells and one expressed in CHOK1 cells. A second TNF-alphavariant BPC1496 was made in a CHO-DG44 cell line. All three samples wereexpressed and purified using mAb Select Sure. In contrast to Example 25,no Superdex column step was carried out. Adalimumab (Product code N00515-01, Lot number 02136XH12) manufactured by Abbott Laboratories, asin Example 25. Adalimumab was formulated in formulations ‘A’ (aspurchased) and ‘B’ (by buffer exchange) as described above in Example25, and the TNF-alpha variants (BPC1494 and 1496) were formulated in‘B’, all at ˜50 mg/mL (total of 5 samples). The samples were filteredwith MillexGV 0.22 um filter under clean laminar flow conditions beforebeing transferred into labelled pre-sterilized glass vials and incubatedat 5° C. and 40° C. for up to 9 weeks. At selected time-points, sampleswere taken and analysed using SEC-HPLC (Table 14), cIEF (Table 15).Other assays as described below were also carried out.

Appearance by Visual Observation.

Samples were observed for clarity under daylight conditions. Bothantibodies in both formulations remained unchanged (clear colourlesssolution) after 9 weeks storage at 5° C. and 40° C.

Protein Concentration (A280 nm) Measurement

Protein concentration was measured using a nanodrop spectrometer, whichis indicative of protein stability. There was no significant differencein the results after 9 weeks storage at 5° C. and 40° C.

Size Exclusion Chromatography (SEC)

SEC-HPLC data (Table 13) showed that adalimumab at 50 mg/ml wasrelatively more stable in formulation ‘B’ compared to formulation ‘A’after storage at 40° C. for 9 weeks. Also, the TNF-alpha variants(BPC1494 and 1496) were relatively as stable or more stable in ‘B’ asadalimumab in B′. No comparison between the variants in ‘A’ and ‘B’ wascarried out.

Note that the Initial TA levels for the TNF-alpha variants wererelatively higher than for adalimumab. Therefore, the results include a% change column at the right hand side to compare the changes fromInitial to Week 9 at 40° C. For example, table 13 shows that after 9week storage, the percentage change in total low molecular weightfragment (TLMWF) in formulation ‘B’ was between 3.82-4.96% compared to6.08% in formulation ‘A’. Similarly, the monomer percentage change informulation ‘A’ was greater for adalimumab than for ‘B’ (7.54 and 4.52%respectively). The TNF-alpha variants in ‘B’ were all relatively atleast as stable or more stable as adalimumab in formulation ‘A’ (%change at Week 9). The results at week 4 for all samples are within the5% TA and/or TLMWF allowance for a commercial product. Therefore, ‘B’has advantages over ‘A’ for both TNF-alpha variants and adalimumab at 50mg/ml.

In particular, the TNF-alpha variant BCP1496 showed a low TLMWF value of3.86 at Week 9 at 40° C.

Capillary Iso-Electric Focusing (c-IEF)

C-IEF data (Table 15) supports the findings in Table 14.

Formulation B shows a reduced % change of % AMI at week 9 for adalimumabas compared to Formulation A (23.53 and 27.57 respectively).

The TNF-alpha variants in ‘B’ are more stable in terms of chargeheterogeneity (i.e. increase in number of peaks) than adalimumab (inboth ‘A’ and ‘B’). For example, at Week 9 there were 5 and 6 peaks foreach of the variants; and 6 and 9 peaks for adalimumab, at 5° C. and 40°C. respectively.

In particular, the TNF-alpha variant BCP1496 and adalimumab, both in‘B’, showed a low % change in % AMI at week 9 of 25.83 and 23.53respectively. The relatively higher % change in % AMI at week 9 for theTNF-alpha variant BCP1496 (CHO DG44) of 38.13 may be due to therelatively high initial % AMI of 75.03.

Functional Binding Assay (ELISA)

The biological activity of adalimumab and the TNF-alpha variants in thetwo formulations was assessed by Biacore. Over the 9 week period ofstorage at 5° C. and 40° C., the samples showed the same % bindingwithin assay variability.

Hence, it can be concluded that formulation ‘B’ can serve as analternative to formulation ‘A’ in a clinical setting withoutcompromising the stability of the antibody at 50 mg/mL dosage strength.

TABLE 14 SEC-HPLC of adalimumab and TNF-alpha variants BPC1494 and 1496in Formulations ‘A’ and ‘B’ at 5° C., 25° C. and 40° C. TLMWF—Total LowMolecular Weight Fragment; Mono—Monomer; TA—Total Aggregate. N = 2Condition Initial Week 1 Week 2 ° C. TA Mono TLMWF TA Mono TLMWF TA MonoTLMWF adalimumab in formulation ‘A’  5° C. 0.30 99.62 0.08 0.30 99.540.16 0.32 99.53 0.15 40° C. 0.39 99.13 0.48 0.51 99.03 0.46 adalimumabin formulation ‘B’  5° C. 0.42 99.45 0.13 0.34 99.49 0.17 0.38 99.450.16 40° C. 0.41 99.36 0.23 0.48 99.23 0.29 TNF-alpha variant BPC1494(CHO DG44) in formulation ‘B’  5° C. 2.76 97.13 0.11 2.65 97.25 0.093.29 96.45 0.25 40° C. 2.96 96.74 0.29 2.98 96.70 0.32 TNF-alpha variantBPC1494 (CHOK1) in formulation ‘B’  5° C. 2.35 97.64 0.00 2.32 97.690.00 2.36 97.64 0.00 40° C. 2.67 97.09 0.24 2.79 96.99 0.22 TNF-alphavariant BPC1496 in formulation ‘B’  5° C. 1.19 98.78 0.04 1.47 98.490.04 1.62 98.34 0.03 40° C. 1.75 97.99 0.26 1.46 98.27 0.27 ConditionWeek 4 Week 9 % Change at Week 9 ° C. TA Mono TLMWF TA Mono TLMWF TAMono TLMWF adalimumab in formulation ‘A’  5° C. 0.29 99.51 0.21 0.3499.49 0.17 1.45 7.54 6.08 40° C. 0.54 98.77 0.69 1.75 92.08 6.16adalimumab in formulation ‘B’  5° C. 0.35 99.42 0.23 0.38 99.44 0.180.46 4.52 4.06 40° C. 0.54 98.85 0.61 0.88 94.93 4.19 TNF-alpha variantBPC1494 (CHO DG44) in formulation ‘B’  5° C. 2.66 97.18 0.16 2.83 97.050.12 1.32 6.28 4.96 40° C. 2.96 96.36 0.68 4.08 90.85 5.07 TNF-alphavariant BPC1494 (CHOK1) in formulation ‘B’  5° C. 2.33 97.67 0.00 2.5697.40 0.04 2.56 7.42 4.88 40° C. 3.17 96.20 0.63 4.91 90.22 4.88TNF-alpha variant BPC1496 in formulation ‘B’  5° C. 1.71 98.15 0.14 1.9297.95 0.13 1.45 5.28 3.82 40° C. 1.40 98.05 0.54 2.64 93.50 3.86

TABLE 15 CE-IEF of adalimumab and TNF-alpha variants BPC1494 and 1496 inFormulations ‘A’ and ‘B’ at 5° C., 25° C. and 40° C. N = 2 Initial Week1 Week 2 Condition % % % ° C. pI R pMI AMI NoP pI R pMI AMI NoP pI R pMIAMI NoP adalimumab in formulation ‘A’  5° C. 8.55-9.01 8.76 58.67 68.56-9.00 8.74 58.28 6 8.57-9.01 8.75 57.42 6 40° C. 8.53-9.01 8.7556.18 6 8.52-9.00 8.75 51.82 6 adalimumab in formulation ‘B’  5° C.8.53-9.02 8.76 57.91 6 8.53-9.01 8.76 59.51 6 8.52-9.01 8.76 56.85 6 40°C. 8.53-9.02 8.76 55.52 6 8.52-9.00 8.75 51.7 6 TNF-alpha variantBPC1494 (CHO DG44) in formulation ‘B’  5° C. 8.22-8.68 8.53 75.03 58.29-8.68 8.52 76.13 5 8.28-8.68 8.52 75.11 5 40° C. 8.22-8.66 8.5170.92 5 8.20-8.67 8.51 64.52 5 TNF-alpha variant BPC1494 (CHOK1) informulation ‘B’  5° C. 8.23-8.68 8.53 63.75 5 8.22-8.68 8.53 63.69 58.22-8.67 8.52 63.37 5 40° C. 8.21-8.66 8.51 60.57 5 8.21-8.65 8.5156.21 5 TNF-alpha variant BPC1496 in formulation ‘B’  5° C. 8.53-8.898.75 65.48 5 8.52-8.88 8.74 64.01 5 8.51-8.88 8.74 67.57 5 40° C.8.51-8.87 8.73 60.52 5 8.51-8.87 8.74 57.82 5 % Week 4 Week 9 ChangeCondition % % Week 9 ° C. pI R pMI AMI NoP pI R pMI AMI NoP % AMIadalimumab in formulation ‘A’  5° C. 8.54-9.01 8.75 59.45 6 8.55-9.028.77 60.37 6 27.57 40° C. 8.53-9.02 8.75 47.24 6 8.36-9.02 8.77 31.10 9adalimumab in formulation ‘B’  5° C. 8.53-9.01 8.75 59.00 6 8.54-9.028.77 59.62 6 23.53 40° C. 8.53-9.01 8.75 47.07 6 8.36-9.02 8.77 34.38 9TNF-alpha variant BPC1494 (CHO DG44) in formulation ‘B’  5° C. 8.28-8.688.52 73.76 5 8.24-8.69 8.53 75.17 5 38.13 40° C. 8.23-8.68 8.53 58.40 58.06-8.69 8.54 36.9 6 TNF-alpha variant BPC1494 (CHOK1) in formulation‘B’  5° C. 8.22-8.68 8.52 62.32 5 8.24-8.70 8.53 63.88 5 33.09 40° C.8.22-8.67 8.52 50.64 5 8.06-8.68 8.54 30.66 6 TNF-alpha variant BPC1496in formulation ‘B’  5° C. 8.52-8.88 8.75 67.06 5 8.53-8.89 8.76 68.75 525.83 40° C. 8.51-8.87 8.74 51.37 5 8.36-8.88 8.76 39.65 6 NT = NotTested; pI R—Pi Range; pMI—pI of Main Isoform; % AMI—% Area MainIsoform; NoP—Number of Peaks.

Example 27 Plasma Concentrations of BPC1494 Following SubcutaneousAdministration in the Male Cynomolgus Monkey

In a repeat dose pharmacokinetic study BPC1494 was administeredsub-cutaneously weekly or biweekly for 4 weeks at 30 or 100 mg/kg tomale cynomolgus monkeys. For group 2 (n=3), the animals wereadministered 2×30 mg/kg doses on day 1 (approximately 1 hour apart)followed by a single 30 mg/kg dose on days 8, 15 and 22. For group 3(n=3), the animals were administered with 2×30 mg/kg doses on day 1(approximately 1 hour apart) followed by a single 30 mg/kg dose on day15. For group 4 (n=3), the animals were administered with 2×100 mg/kgdoses on day 1 (approximately 1 hour apart) followed by a single 100mg/kg dose on day 15. Plasma samples were taken at intervals throughoutthe dosing and recovery phases of the study.

Plasma samples were analyzed for BPC1494 using a qualified analyticalmethod based on sample dilution followed by immunoassay analysis Plasmasamples were analyzed for BPC1494 or BPC1492. The method used 10 μg/mlbiotinylated recombinant human TNF-alpha as the capture antigen and a1:100 dilution of AlexaFluor 647-labelled anti-human IgG (Fc specific)antibody as the detection antibody (G18-145). The lower limit ofquantification (LLQ) for BPC1494 was 1 μg/mL using a 50 μL aliquot of100-fold diluted monkey plasma with a higher limit of quantification(HLQ) of 100 μg/mL. The computer systems that were used on this study toacquire and quantify data included Gyrolab Workstation Version 5.2.0,Gyrolab Companion version 1.0 and SMS2000 version 2.3. PK analysis wasperformed by non-compartmental pharmacokinetic analysis using WinNonlinEnterprise Pheonix version 6.1.

Pharmacokinetic data is presented in Table 16 with parameters determinedfrom last dose received on Week 4 to the time point (t) 840 hours postdosing for 30 mg/kg/week dose group (2) and last dose received on Week 3to the time point (t) 1008 hours post dosing for 30 & 100 mg/kg/biweeklydose groups (3 and 4).

TABLE 16 Individual and Mean Pharmacokinetic Parameters for BPC1494 inthe Male Cynomolgus Monkey Following Subcutaneous Dosing of BPC1494 at30 mg/kg/week or 30 and 100 mg/kg/biweekly over a 4-Week InvestigativeStudy Pharmacokinetic Parameters b Dose Median Estimated c Estimated c(mg/kg/ Animal AUC0-t Cmax Tmax t½ MRT CL_F Vz_F biweekly) Number (mg ·h/mL) (mg/mL) (h) (h) (h) (mL/h/kg) (mL/kg)  30a P12M-272 923 1.51 168 616 367 0.125 111   P12M-273 758 1.29 168  604 368 0.141 123   P12M-274d  21.3 0.135 24 226 142 3.01 978   Mean 841 1.40 168  610 367 0.133117   (568) (0.977) (482) (292) (1.09) (404)   30 P12M-275 743 1.08 24420 419 0.115 69.5 P12M-276 538 2.31 48 197 307 0.141 40.2 P12M-277d 2391.09 24 123 189 0.217 38.6 Mean 641 1.70 36 309 363 0.128 54.9 (507)(1.49) (24) (247) (305) (0.158) (49.4) 100  P12M-278 2760  5.89 24 398374 0.0998 57.3 P12M-279 2480  5.21 72 332 362 0.131 62.9 P12M-280 2080 4.10 72 331 364 0.123 58.8 Mean 2440  5.07 72 354 367 0.118 59.7 aGroup2 animals received 30 mg/kg weekly for 4 weeks b) Pharmacokineticparameters determined from last dose received on Week 4 to the timepoint (t) 840 hours post dosing for 30 mg/kg/week and last dose receivedon Week 3 to the time point (t)1008 hours post dosing for 30 & 100mg/kg/biweekly c) Cl_F and Vz_F are estimates due to elimination phasefollowing multiple doses and steady state not yet achieved. Parameterestimates have been calculated from i) using AUC0-168 or 336, ii)extrapolation of data from week 1 based on half-life and iii) usingtotal dose over the defined sampling with AUC0-inf dAnimal 274 and 277excluded from mean pharmacokinetic calculations based on scientificjudgment that these animals are likely to be exhibiting an anti-drugantibody response. Mean data shown in parentheses are inclusive of theseanimals.

Example 28 SPR Binding Analysis of FcRn to Protein L Captured Anti-TNFαmAbs

The study was carried out using the ProteOn™ XPR36 (BioRad™) biosensormachine, a surface plasmon based machine designed for label freekinetic/affinity measurements. Protein L was immobilised on a GLM chip(BioRad, Cat No: 176-5012) by primary amine coupling. This surface wasthen used to capture the humanised antibodies, human and cyno FcRn (bothin-house materials) was then used as analytes at 2048 nM, 512 nM, 128nM, 32 nM, and 8 nM, an injection of buffer alone (i.e. 0 nM) used todouble reference the binding curves. Regeneration of the protein Lsurface was carried out using Glycine-HCl pH1.5. The assay was run at25° C. and run in HBS-EP pH7.4 and HBS-EP pH6.0 with human or cynomolgusFcRn diluted in appropriate buffer. Affinities were calculated using theEquilibrium model, inherent to the PrateOn analysis software, using a“Global R-max” for binding at pH6.0 and the R-max from binding at pH6.0for affinity calculation at pH7.4. Since the binding curves did notreach saturation at pH7.4, the values obtained are unlikely to be trueaffinities however were used to rank the binding of the antibodiestested.

The binding affinity of different batches of BPC1492, BPC1494 andBPC1496 for human FcRn was compared using antibodies captures by ProteinL. Table 17 shows the results from a series of experiments using thisformat. The data confirms that BPC1494 and BPC1496 have an improvedaffinity for recombinant human FcRn compared to BPC1492 at both pH6.0and pH7.4. The fold improvement in binding affinity of BPC1494 for FcRncompared to BPC1492 differs from experiment to experiment due to changesin the Protein L activity on the capture. However, in the experimentsshown in Table 17, the fold improvement in binding affinity at pH6.0ranges between 3.5-fold and 16.3-fold. It was not possible to determinethe fold improvement in binding affinity at pH7.4 due to the weakbinding activity of human IgG for FcRn at neutral pH.

The binding affinity of different batches of BPC1492, BPC1494 andBPC1496 for cynomolgus FcRn was also compared using antibodies capturedwith Protein L. Table 18 shows the results from the experiment usingthis format. The data confirms that BPC1494 has an improved affinity forrecombinant cynomolgus FcRn compared to BPC1492 at both pH6.0 and pH7.4.The fold improvement in binding affinity of BPC1494 (range 41.8-46.8 nM)for cynomolgus FcRn compared to BPC1492 (range 394-398 nM) isapproximately 9-fold at pH6. It was not possible to determine the foldimprovement in binding affinity at pH7.4 due to the weak bindingactivity of BPC1492 for FcRn.

TABLE 17 Recombinant human FcRn binding affinities using the Protein Lcapture method Affinity KD (nM) BPC1492 BPC1494 BPC1496 Batch BatchBatch HEK HEK CHO clinical HEK HEK GRITS HEK HEK GRITS Expt. pH 14061348 grade 1407 1350 42954 1352 1408 42955 5 6 320.0  325.0  315.0      6.08** 24.9 26.2 14.3 16.9 15.4 7.4 NAB NAB NAB  2020** 12600 117008980 9830 9670 4 6 50.9 54.8 55.5     1.33 4.05 4.50 2.35 3.60 2.33 7.4NAB NAB NAB  303 5270 4740 6820 7550 7550 3 6 16.0 16.8 17.3     0.7011.960 2.430 2.200 4.140 1.810 7.4 NAB NAB NAB 1760 10500 10900 7830 80508460 2 6 13.1 12.9 13.9 ## 0.359 0.979 0.978 2.440 0.546 7.4 NAB NAB NAB2010 9190 9330 10900 9480 9550 1 6 ND 234   ND ND 66 ND ND 85 ND 7.4 NDNAB ND ND NAB ND ND 2010 ND **although data points have been reported,the values should be treated with caution because these data are notconsistent with the data obtained for the other batches of the samemolecule during this experiment NAB = no analysable binding ND = nottested in this experiment ## = high affinity binding - beyond thesensitivity of the machine

TABLE 18 Recombinant cynomolgus FcRn binding affinities using theProtein L capture method pH 6 pH 7.4 Batch number Construct KD (nM) KD(nM) GRITS44463 BPC1494 46.8 14800 MCB16Marc2012 BPC1494 41.8 13300GRITS42954 BPC1494 43.2 13700 Clinical grade BPC1492 394 No bindingGRITS44348 BPC1492 398 No binding

TABLE A Sequence identifier (SEQ ID NO) Poly- Amino Descriptionnucleotide acid Anti-TNF antibody light chain 1 2 Anti-TNF antibodyvariable domain (VL) — 3 anti-TNF antibody heavy chain plus 4 5M252Y/S254T/T256E modification Anti-TNF antibody heavy variable domain(VH) — 6 IgG1 constant domain plus — 7 M252Y/S254T/T256E modificationAnti-TNF antibody heavy chain plus 8 9 M428L/N434S modification IgG1constant domain plus M428L/N434S — 10 modification Anti-TNF antibodyheavy chain (wild-type IgG1) 11 12 IgG1 constant domain (wild-type) — 13Anti-TNF antibody heavy chain plus 14 15 T250Q/M428L modification IgG1constant domain plus T250Q/M428L — 16 modification Anti-TNF antibodyheavy chain plus V308F 17 18 modification IgG1 constant domain plusV308F modification — 19 Anti-TNF antibody heavy chain plus V259I 20 21modification IgG1 constant domain plus V259I modification — 22 Anti-TNFantibody heavy chain plus P257L and 23 24 N434Y variant IgG1 constantdomain plus P257L and N434Y — 25 modification Signal peptide sequence —26 Anti-TNF antibody CDRH1 — 27 Anti-TNF antibody CDRH2 — 28 Anti-TNFantibody CDRH3 — 29 Anti-TNF antibody CDRL1 — 30 Anti-TNF antibody CDRL2— 31 Anti-TNF antibody CDRL3 — 32 Anti-TNF antibody CDRH1 variant —33-38 Cimzia (certolizumab) LC (VL + Ck 39 Anti-TNF antibody CDRH3variant — 40-49 Anti-TNF antibody CDRL1 variant — 50-61 Anti-TNFantibody CDRL2 variant — 62-72 Anti-TNF antibody CDRL3 variant — 73-76cb1-3-VH 77 78 cb2-44-VH 79 80 cb1-39-VH 81 82 cb1-31-VH 83 84 cb2-11-VH85 86 cb2-40-VH 87 88 cb2-35-VH 89 90 cb2-28-VH 91 92 cb2-38-VH 93 94cb2-20-VH 95 96 cb1-8-VL 97 98 cb1-43-VL 99 100 cb1-45-VL 101 102cb1-4-VL 103 104 cb1-41-VL 105 106 cb1-37-VL 107 108 cb1-39-VL 109 110cb1-33-VL 111 112 cb1-35-VL 113 114 cb1-31-VL 115 116 cb1-29-VL 117 118cb1-22-VL 119 120 cb1-23-VL 121 122 cb1-12-VL 123 124 cb1-10-VL 125 126cb2-1-VL 127 128 cb2-11-VL 129 130 cb2-40-VL 131 132 cb2-35-VL 133 134cb2-28-VL 135 136 cb2-20-VL 137 138 cb1-3-VL 139 140 cb2-6-VL 141 142cb2-44-VL 143 144 Anti-TNF antibody heavy chain variant cb1-3-VH — 145plus M252Y/S254T/T256E modification Anti-TNF antibody heavy chainvariant cb2-44- — 146 VH plus M252Y/S254T/T256E modification Anti-TNFantibody light chain variant cb1-3-VL 147 148 Anti-TNF antibody lightchain variant cb2-6-VL 149 150 Anti-TNF antibody light chain variantcb2-44-VL 151 152 Anti-TNF antibody heavy chain variant cb1-3-VH 153 154Anti-TNF antibody heavy chain variant cb2-44- 155 156 VH Pascolizumabheavy chain containing the 157 158 M252Y/S254T/T256E modificationsPascolizumab light chain 159 160 Pascolizumab heavy chain — 161Alternative anti-TNF antibody heavy chain plus 162 M428L/N434Smodification Alternative IgG1 constant domain plus 163 M428L/N434Smodification Anti-TNF antibody heavy chain plus 164 H433K/N434Fmodification IgG1 constant domain plus H433K/N434F 165 modificationAlternative anti-TNF antibody heavy chain plus 166 H433K/N434Fmodification Alternative IgG1 constant domain plus 167 H433K/N434Fmodification Alternative anti-TNF antibody heavy chain plus 168M428L/N434S modification Alternative IgG1/2 constant domain plus 169M428L/N434S modification Golimumab_VH 170 Golimumab_VL 171 Golimumab HC172 Golimumab LC 173 Remicade VH 174 Remicade VL 175 Remicade HC 176Remicade LC 177 Cimzia (certolizumab) VH 178 Cimzia (certolizumab) VL179 Cimzia (certolizumab) HC (VH + CH1) 180

Sequence listing SEQ ID NO: 1Polynucleotide sequence of the anti-TNF antibody light chainGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCGGGCCAGCCAGGGCATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCACCCTGCAGAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACAACAGAGCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTCAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAAGTGCAGTGGAAAGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 2Protein sequence of the anti-TNF antibody light chainDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 3Protein sequence of the anti-TNF antibody variable domain (VL)DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRT SEQ ID NO: 4Polynucleotide sequence of the anti-TNF antibodyheavy chain plus M252Y/S254T/T256E modificationGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGTACATCACCAGAGAGCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 5Protein sequence of the anti-TNF antibody heavy chainplus M252Y/S254T/T256E modificationEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSEQ ID NO: 6 Protein sequence of the anti-TNF antibody heavyvariable domain (VH)EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS SEQ ID NO: 7Protein sequence of the IgG1 constant domain plusM252Y/S254T/T256E modificationASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 8 Polynucleotide sequence of the anti-TNF antibodyheavy chain plus M428L/N434S modificationGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGCTGCACGAGGCCCTGCACAGCCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 9Protein sequence of the anti-TNF antibody heavychain plus M428L/N434S modificationEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSL SPGKSEQ ID NO: 10 Protein sequence of the IgG1 constant domain plusM428L/N434S modificationASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK SEQ ID NO: 11 Polynucleotide sequence of the anti-TNF antibodyheavy chain (wild-type IgG1)GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 12Protein sequence of the anti-TNF antibody heavy chain (wild-type IgG1)EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSEQ ID NO: 13 Protein sequence of the IgG1 constant domain (wild-type)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 14 Polynucleotide sequence of the anti-TNF antibodyheavy chain plus T250Q/M428L modificationGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACcaaCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGtTGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 15Protein sequence of the anti-TNF antibody heavychain plus T250Q/M428L modificationEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSL SPGKSEQ ID NO: 16 Protein sequence of the IgG1 constant domain plusT250Q/M428L modificationASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK SEQ ID NO: 17 Polynucleotide sequence of the anti-TNF antibodyheavy chain plus V308F modificationGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCtTcCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 18Protein sequence of the anti-TNF antibody heavychain plus V308F modificationEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTFLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSEQ ID NO: 19 Protein sequence of the IgG1 constant domains plusV308F modificationASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTFLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 20 Polynucleotide sequence of the anti-TNF antibodyheavy chain plus V259I modificationGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGATCACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAAGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 21Protein sequence of the anti-TNF antibody heavychain plus V259I modificationEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEITCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSEQ ID NO: 22 Protein sequence of the IgG1 constant domainsplus V259I modificationASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEITCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 23 Polynucleotide sequence of the anti-TNF antibodyheavy chain plus P257L and N434Y variantGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCTGGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACTATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 24Protein sequence of the anti-TNF antibody heavychain plus P257L and N434Y modificationEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTLEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSL SPGKSEQ ID NO: 25 Protein sequence of the IgG1 constant domains plusP257L and N434Y modificationASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTLEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSLSPGK SEQ ID NO: 26 Signal peptide sequence MGWSCIILFLVATATGVHSSEQ ID NO: 27 anti-TNF antibody CDRH1 DYAMH SEQ ID NO: 28anti-TNF antibody CDRH2 AITWNSGHIDYADSVEG SEQ ID NO: 29anti-TNF antibody CDRH3 VSYLSTASSLDY SEQ ID NO: 30anti-TNF antibody CDRL1 RASQGIRNYLA SEQ ID NO: 31anti-TNF antibody CDRL2 AASTLQS SEQ ID NO: 32 anti-TNF antibody CDRL3QRYNRAPYT SEQ ID NO: 33 anti-TNF antibody CDRH1 variant QYAMHSEQ ID NO: 34 anti-TNF antibody CDRH1 variant HYALH SEQ ID NO: 35anti-TNF antibody CDRH1 variant HYAMH SEQ ID NO: 36anti-TNF antibody CDRH1 variant QHALH SEQ ID NO: 37anti-TNF antibody CDRH1 variant QHAMH SEQ ID NO: 38anti-TNF antibody CDRH1 variant DHALH SEQ ID NO: 39Cimzia (certolizumab) LC (VL + Ck)DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASFLYSGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 40 anti-TNF antibody CDRH3 variantVHYLSTASQLHH SEQ ID NO: 41 anti-TNF antibody CDRH3 variant VQYLSTASSLQSSEQ ID NO: 42 anti-TNF antibody CDRH3 variant VKYLSTASSLHY SEQ ID NO: 43anti-TNF antibody CDRH3 variant VKYLSTASNLES SEQ ID NO: 44anti-TNF antibody CDRH3 variant VHYLSTASSLDY SEQ ID NO: 45anti-TNF antibody CDRH3 variant VSYLSTASSLQS SEQ ID NO: 46anti-TNF antibody CDRH3 variant VRYLSTASNLQH SEQ ID NO: 47anti-TNF antibody CDRH3 variant VQYLSTASQLHS SEQ ID NO: 48anti-TNF antibody CDRH3 variant VRYLSTASQLDY SEQ ID NO: 49anti-TNF antibody CDRH3 variant VRYLSTASSLDY SEQ ID NO: 50anti-TNF antibody CDRL1 variant HASKKIRNYLA SEQ ID NO: 51anti-TNF antibody CDRL1 variant HASRKLRNYLA SEQ ID NO: 52anti-TNF antibody CDRL1 variant HASRRLRNYLA SEQ ID NO: 53anti-TNF antibody CDRL1 variant HASKRIRNYLA SEQ ID NO: 54anti-TNF antibody CDRL1 variant HASRKIRNYLA SEQ ID NO: 55anti-TNF antibody CDRL1 variant HASRRIRNYLA SEQ ID NO: 56anti-TNF antibody CDRL1 variant HASREIRNYLA SEQ ID NO: 57anti-TNF antibody CDRL1 variant HASQGIRNYLA SEQ ID NO: 58anti-TNF antibody CDRL1 variant HASQKIRNYLA SEQ ID NO: 59anti-TNF antibody CDRL1 variant RASRGLRNYLA SEQ ID NO: 60anti-TNF antibody CDRL1 variant HASQRIRNYLA SEQ ID NO: 61anti-TNF antibody CDRL1 variant RASRRIRNYLA SEQ ID NO: 62anti-TNF antibody CDRL2 variant AASSLLR SEQ ID NO: 63anti-TNF antibody CDRL2 variant AASSLLK SEQ ID NO: 64anti-TNF antibody CDRL2 variant AASSLLP SEQ ID NO: 65anti-TNF antibody CDRL2 variant AASSLQP SEQ ID NO: 66anti-TNF antibody CDRL2 variant AASSLLH SEQ ID NO: 67anti-TNF antibody CDRL2 variant AASSFLP SEQ ID NO: 68anti-TNF antibody CDRL2 variant AASSLLQ SEQ ID NO: 69anti-TNF antibody CDRL2 variant AASSLQQ SEQ ID NO: 70anti-TNF antibody CDRL2 variant AASTLLK SEQ ID NO: 71anti-TNF antibody CDRL2 variant AASSLQN SEQ ID NO: 72anti-TNF antibody CDRL2 variant AASSLQK SEQ ID NO: 73anti-TNF antibody CDRL3 variant QRYDRPPYT SEQ ID NO: 74anti-TNF antibody CDRL3 variant QRYDKPPYT SEQ ID NO: 75anti-TNF antibody CDRL3 variant QRYNRPPYT SEQ ID NO: 76anti-TNF antibody CDRL3 variant QRYNKPPYT SEQ ID NO: 77Polynucleotide sequence of anti-TNF antibodyvariable heavy domain variant cb1-3-VH (aka cb2-6-VH)GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCSEQ ID NO: 78 Protein sequence of anti-TNF antibody variableheavy domain variant cb1-3-VH (aka cb2-6-VH)EVQLVESGGGLVQPGRSLRLSCAASGFTFDQYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSSEQ ID NO: 79 Polynucleotide sequence of anti-TNF antibodyvariable heavy domain variant cb2-44-VHGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACCACGCCCTGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGAGGTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTC CAGCSEQ ID NO: 80 Protein sequence of anti-TNF antibody variableheavy domain variant cb2-44-VHEVQLVESGGGLVQPGRSLRLSCAASGFTFDDHALHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVRYLSTASSLDYWGQGTLVTVSS SEQ ID NO: 81Polynucleotide sequence of anti-TNF antibodyvariable heavy domain variant cb1-39-VHGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACCACTACGCCCTGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCSEQ ID NO: 82 Protein sequence of anti-TNF antibody variableheavy domain variant cb1-39-VHEVQLVESGGGLVQPGRSLRLSCAASGFTFDHYALHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS SEQ ID NO: 83Polynucleotide sequence of anti-TNF antibodyvariable heavy domain variant cb1-31-VHGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGCACTACCTGAGCACCGCCAGCCAACTGCACCACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCSEQ ID NO: 84 Protein sequence of anti-TNF antibody variableheavy domain variant cb1-31-VHEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVHYLSTASQLHHWGQGTLVTVSSSEQ ID NO: 85 Polynucleotide sequence of anti-TNF antibodyvariable heavy domain variant cb2-11-VHGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACCACTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGCAGTACCTGAGCACCGCCAGCAGCCTGCAGAGCTGGGGCCAGGGCACACTAGTGACCGTGTC CAGCSEQ ID NO: 86 Protein sequence of anti-TNF antibody variableheavy domain variant cb2-11-VHEVQLVESGGGLVQPGRSLRLSCAASGFTFDHYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVQYLSTASSLQSWGQGTLVTVSSSEQ ID NO: 87 Polynucleotide sequence of anti-TNF antibodyvariable heavy domain variant cb2-40-VHGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGAAGTACCTGAGCACCGCCAGCAGCCTGCACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCSEQ ID NO: 88 Protein sequence of anti-TNF antibody variableheavy domain variant cb2-40-VHEVQLVESGGGLVQPGRSLRLSCAASGFTFDQYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVKYLSTASSLHYWGQGTLVTVSSSEQ ID NO: 89 Polynucleotide sequence of anti-TNF antibodyvariable heavy domain variant cb2-35-VHGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGCACGCCCTGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGCACTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCSEQ ID NO: 90 Protein sequence of anti-TNF antibody variableheavy domain variant cb2-35-VHEVQLVESGGGLVQPGRSLRLSCAASGFTFDQHALHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVHYLSTASSLDYWGQGTLVTVSS SEQ ID NO: 91Polynucleotide sequence of anti-TNF antibodyvariable heavy domain variant cb2-28-VHGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGCACTACCTGAGCACCGCCAGCCAGCTGCACCACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCSEQ ID NO: 92 Protein sequence of anti-TNF antibody variableheavy domain variant cb2-28-VHEVQLVESGGGLVQPGRSLRLSCAASGFTFDQYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVHYLSTASQLHHWGQGTLVTVSSSEQ ID NO: 93 Polynucleotide sequence of anti-TNF antibodyvariable heavy domain variant cb2-38-VHGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGCACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCSEQ ID NO: 94 Protein sequence of anti-TNF antibody variableheavy domain variant cb2-38-VHEVQLVESGGGLVQPGRSLRLSCAASGFTFDQHAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSSEQ ID NO: 95 Polynucleotide sequence of anti-TNF antibodyvariable heavy domain variant cb2-20-VHGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGAAGTACCTGAGCACCGCCAGCAACCTGGAGAGCTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCSEQ ID NO: 96 Protein sequence of anti-TNF antibody variableheavy domain variant cb2-20-VHEVQLVESGGGLVQPGRSLRLSCAASGFTFDQYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVKYLSTASNLESWGQGTLVTVSSSEQ ID NO: 97 Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-8-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAAGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAGGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 98Protein sequence of anti-TNF antibody variablelight domain variant cb1-8-VLDIQMTQSPSSLSASVGDRVTITCHASKKIRNYLAWYQQKPGKAPKLLIYAASSLLRGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 99Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-43-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAAGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAAGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 100Protein sequence of anti-TNF antibody variablelight domain variant cb1-43-VLDIQMTQSPSSLSASVGDRVTITCHASRKLRNYLAWYQQKPGKAPKLLIYAASSLLKGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 101Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-45-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAGGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCCCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 102Protein sequence of anti-TNF antibody variablelight domain variant cb1-45-VLDIQMTQSPSSLSASVGDRVTITCHASRRLRNYLAWYQQKPGKAPKLLIYAASSLLPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 103Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-4-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAAGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAGGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 104Protein sequence of anti-TNF antibody variablelight domain variant cb1-4-VLDIQMTQSPSSLSASVGDRVTITCHASRKLRNYLAWYQQKPGKAPKLLIYAASSLLRGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 105Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-41-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAAGAGGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAAGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAAGCCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 106Protein sequence of anti-TNF antibody variablelight domain variant cb1-41-VLDIQMTQSPSSLSASVGDRVTITCHASKRIRNYLAWYQQKPGKAPKLLIYAASSLLKGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDKPPYTFGQGTKVEIKRT SEQ ID NO: 107Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-37-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAAGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAGGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACAACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 108Protein sequence of anti-TNF antibody variablelight domain variant cb1-37-VLDIQMTQSPSSLSASVGDRVTITCHASRKLRNYLAWYQQKPGKAPKLLIYAASSLLRGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRPPYTFGQGTKVEIKRT SEQ ID NO: 109Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-39-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGCCCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 110Protein sequence of anti-TNF antibody variablelight domain variant cb1-39-VLDIQMTQSPSSLSASVGDRVTITCHASRKIRNYLAWYQQKPGKAPKLLIYAASSLQPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 111Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-33-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAGGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCACGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 112Protein sequence of anti-TNF antibody variablelight domain variant cb1-33-VLDIQMTQSPSSLSASVGDRVTITCHASRRIRNYLAWYQQKPGKAPKLLIYAASSLLHGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 113Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-35-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAGGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGCCCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 114Protein sequence of anti-TNF antibody variablelight domain variant cb1-35-VLDIQMTQSPSSLSASVGDRVTITCHASRRLRNYLAWYQQKPGKAPKLLIYAASSLQPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 115Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-31-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAGGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAAGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACAACAAGCCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 116Protein sequence of anti-TNF antibody variablelight domain variant cb1-31-VLDIQMTQSPSSLSASVGDRVTITCHASRRLRNYLAWYQQKPGKAPKLLIYAASSLLKGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNKPPYTFGQGTKVEIKRT SEQ ID NO: 117Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-29-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCTTCCTGCCCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 118Protein sequence of anti-TNF antibody variablelight domain variant cb1-29-VLDIQMTQSPSSLSASVGDRVTITCHASRKIRNYLAWYQQKPGKAPKLLIYAASSFLPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 119Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-22-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAAGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGCCCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 120Protein sequence of anti-TNF antibody variablelight domain variant cb1-22-VLDIQMTQSPSSLSASVGDRVTITCHASKKIRNYLAWYQQKPGKAPKLLIYAASSLQPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 121Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-23-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAGGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCAGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 122Protein sequence of anti-TNF antibody variablelight domain variant cb1-23-VLDIQMTQSPSSLSASVGDRVTITCHASRRIRNYLAWYQQKPGKAPKLLIYAASSLLQGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 123Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-12-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAAGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGCAGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 124Protein sequence of anti-TNF antibody variablelight domain variant cb1-12-VLDIQMTQSPSSLSASVGDRVTITCHASRKLRNYLAWYQQKPGKAPKLLIYAASSLQQGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 125Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-10-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAAGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCCCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 126Protein sequence of anti-TNF antibody variablelight domain variant cb1-10-VLDIQMTQSPSSLSASVGDRVTITCHASRKLRNYLAWYQQKPGKAPKLLIYAASSLLPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 127Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb2-1-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGGAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCCCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 128Protein sequence of anti-TNF antibody variablelight domain variant cb2-1-VLDIQMTQSPSSLSASVGDRVTITCHASREIRNYLAWYQQKPGKAPKLLIYAASSLLPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 129Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb2-11-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCCAGGGCATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCACCCTGCTGAAGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 130Protein sequence of anti-TNF antibody variablelight domain variant cb2-11-VLDIQMTQSPSSLSASVGDRVTITCHASQGIRNYLAWYQQKPGKAPKLLIYAASTLLKGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 131Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb2-40-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCCAGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGCAGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 132Protein sequence of anti-TNF antibody variablelight domain variant cb2-40-VLDIQMTQSPSSLSASVGDRVTITCHASQKIRNYLAWYQQKPGKAPKLLIYAASSLQQGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 133Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb2-35-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAGGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCACGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 134Protein sequence of anti-TNF antibody variablelight domain variant cb2-35-VLDIQMTQSPSSLSASVGDRVTITCHASRRLRNYLAWYQQKPGKAPKLLIYAASSLLHGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 135Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb2-28-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAGGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAAGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 136Protein sequence of anti-TNF antibody variablelight domain variant cb2-28-VLDIQMTQSPSSLSASVGDRVTITCHASRRLRNYLAWYQQKPGKAPKLLIYAASSLLKGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 137Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb2-20-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAAGAGGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAGGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACAACAAGCCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 138Protein sequence of anti-TNF antibody variablelight domain variant cb2-20-VLDIQMTQSPSSLSASVGDRVTITCHASKRIRNYLAWYQQKPGKAPKLLIYAASSLLRGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNKPPYTFGQGTKVEIKRT SEQ ID NO: 139Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb1-3-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAGGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAAGCCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 140Protein sequence of anti-TNF antibody variablelight domain variant cb1-3-VLDIQMTQSPSSLSASVGDRVTITCHASRKIRNYLAWYQQKPGKAPKLLIYAASSLLRGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDKPPYTFGQGTKVEIKRT SEQ ID NO: 141Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb2-6-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAAGAGGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAAGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACAACAAGCCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 142Protein sequence of anti-TNF antibody variablelight domain variant cb2-6-VLDIQMTQSPSSLSASVGDRVTITCHASKRIRNYLAWYQQKPGKAPKLLIYAASSLLKGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNKPPYTFGQGTKVEIKRT SEQ ID NO: 143Polynucleotide sequence of anti-TNF antibodyvariable light domain variant cb2-44-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCCCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 144Protein sequence of anti-TNF antibody variablelight domain variant cb2-44-VLDIQMTQSPSSLSASVGDRVTITCHASRKIRNYLAWYQQKPGKAPKLLIYAASSLLPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 145Protein sequence of anti-TNF antibody heavy chainvariant cb1-3-VH plus M252Y/S254T/T256E modificationEVQLVESGGGLVQPGRSLRLSCAASGFTFDQYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSEQ ID NO: 146 Protein sequence of anti-TNF antibody heavy chainvariant cb2-44-VH plus M252Y/S254T/T256E modificationEVQLVESGGGLVQPGRSLRLSCAASGFTFDDHALHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVRYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSEQ ID NO: 147 Polynucleotide sequence of anti-TNF antibody lightchain variant cb1-3-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAGGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAAGCCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 148Protein sequence of anti-TNF antibody light chain variant cb1-3-VLDIQMTQSPSSLSASVGDRVTITCHASRKIRNYLAWYQQKPGKAPKLLIYAASSLLRGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDKPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 149Polynucleotide sequence of anti-TNF antibody lightchain variant cb2-6-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAAGAGGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAAGGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACAACAAGCCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 150Protein sequence of anti-TNF antibody light chain variant cb2-6-VLDIQMTQSPSSLSASVGDRVTITCHASKRIRNYLAWYQQKPGKAPKLLIYAASSLLKGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNKPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 151Polynucleotide sequence of anti-TNF antibody lightchain variant cb2-44-VLGATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCATCACCTGCCACGCCAGCAGGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCCCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 152Protein sequence of anti-TNF antibody light chain variant cb2-44-VLDIQMTQSPSSLSASVGDRVTITCHASRKIRNYLAWYQQKPGKAPKLLIYAASSLLPGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 153Polynucleotide sequence of anti-TNF antibody heavychain variant cb1-3-VHGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGTACGCCATGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCCTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 154Protein sequence of anti-TNF antibody heavy chain variant cb1-3-VHEVQLVESGGGLVQPGRSLRLSCAASGFTFDQYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSEQ ID NO: 155 Polynucleotide sequence of anti-TNF antibody heavychain variant cb2-44-VHGAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACCACGCCCTGCACTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTACGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGAGGTACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 156Protein sequence of anti-TNF antibody heavy chain variant cb2-44-VHEVQLVESGGGLVQPGRSLRLSCAASGFTFDDHALHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVRYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSEQ ID NO: 157 Polynucleotide sequence of pascolizumab heavy chaincontaining the M252Y/S254T/T256E modificationsCAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGCAGCCACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGTACAACCCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGGTGCTGACCATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGAGACCGTCTTCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGtacATCacCAGAgagCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 158Protein sequence of pascolizumab heavy chaincontaining the M252Y/S254T/T256E modificationsQVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKSEQ ID NO: 159 Polynucleotide sequence of pascolizumab light chainGACATCGTGCTGACCCAGAGCCCCTCTTCCCTGAGCGCAAGCGTGGGCGATAGGGTGACCATCACCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACATGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCAACCTCGAGTCAGGCATTCCCAGCAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCTTCACAATCAGCAGCCTGCAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGAGCAACGAGGACCCTCCCACCTTCGGACAGGGCACCAAGGTCGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 160Protein sequence of pascolizumab light chainDIVLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLESGIPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSNEDPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 161Protein sequence of pascolizumab heavy chainQVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVVLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKSEQ ID NO: 162 Alternative protein sequence of the anti-TNF antibodyheavy chain plus M428L/N434S modificationEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLS LSPGKSEQ ID NO: 163 Alternative protein sequence of the IgG1 constantdomain plus M428L/N434S modificationASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK SEQ ID NO: 164Protein sequence of the anti-TNF antibody heavychain plus H433K/N434F modificationEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSL SPGKSEQ ID NO: 165 Protein sequence of the IgG1 constant domain plusH433K/N434F modificationASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK SEQ ID NO: 166Alternative protein sequence of the anti-TNF antibodyheavy chain plus H433K/N434F modificationEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLS LSPGKSEQ ID NO: 167 Alternative protein sequence of the IgG1 constantdomain plus H433K/N434F modificationASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK SEQ ID NO: 168Alternative protein sequence of the anti-TNF antibodyheavy chain plus M428L/N434S modificationEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLS LSPGKSEQ ID NO: 169 Alternative protein sequence of the IgG1/2 constantdomain plus M428L/N434S modificationASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK SEQ ID NO: 170 Golimumab_VHQVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVS SSEQ ID NO: 171 Golimumab_VLEIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKRT SEQ ID NO: 172Golimumab_HCQVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 173 Golimumab_LCEIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 174 Remicade_VHEVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNYYGSTYDYWGQGTTLTVSS SEQ ID NO: 175Remicade_VLDILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVKRT SEQ ID NO: 176 Remicade_HCEVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNYYGSTYDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSEQ ID NO: 177 Remicade_LCDILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 178 Cimzia (certolizumab) VHEVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMGWINTYIGEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRSYAMDYWGQGTLVTVSS SEQ ID NO: 179Cimzia (certolizumab) VLDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASFLYSGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKVEIKRT SEQ ID NO: 180Cimzia (certolizumab) HC (VH + CH1)EVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMGWINTYIGEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCAA

1-44. (canceled)
 45. An antibody comprising heavy and light chainshaving polypeptide sequences of SEQ ID NO:5 and SEQ ID NO:2,respectively.
 46. A method of treating a human patient with rheumatoidarthritis, polyarticular juvenile idiopathic arthritis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease or psoriasiscomprising the step of administering the antibody of claim 45.